The first known inhabitants were Algonquian-speaking Native Americans, of the Setauket and Unkechaug tribes. The first English settlers arrived around 1640. The latter founding year was recognized in 1976, when the Brookhaven Town Bicentennial Commission proposed setting the date on the seal to 1655 in line with this first deed of settlement of the Town of Brookhaven at Setauket on April 14, 1655. Considering this founding year of 1655, Brookhaven is the fifth English township on Long Island following Southampton, Southold, Huntington, and East Hampton.

The first English settlement was named Setauket after the Native American tribe. The names "Brookhaven" and "Setauket" were initially used interchangeably to describe the village or the town. The verbal division between the smaller hamlet of Setauket and township of Brookhaven was not set until well into the 19th century. A point of confusion is the existence of the hamlet named Brookhaven, which was in fact named for the township in 1879. Other names used in the settlement's first decades were Ashford, after Ashford, Kent in England, and Cromwell Bay, for English Protestant leader Oliver Cromwell.

The original purchase from the native Setalcott tribe that took place in 1655 encompassed the land making up present-day Setauket, Stony Brook, and Port Jefferson. A second purchase was made by Richard Woodhull in 1664 expanded this tract eastward along the North Shore to additionally include all lands from the Old Mans area (Mount Sinai and Miller Place) to Wading River. Richard Woodhull was the direct heir of Eustace de Vesci, a British noble who was a signator of the Magna Charta. Another land purchase in the same year expanded Brookhaven to the South Shore of Long Island.

Brookhaven was integrated into the Province of New York following that colony's establishment in 1664, and in 1666 Governor Richard Nicolls granted a patent for the town which confirmed title to the lands purchased. Governor Thomas Dongan issued a patent in 1686 which granted powers to the town and established a representative form of government. The town seal was authorized at this time. The central element of the town seal, the letter “D”, was designated to the Town of Brookhaven as its official cattle earmark by the Duke's Laws of 1665. Although no records exist dating to the town seal's original design plan, it is generally thought that the seal's olive branch signified peace and the whaling tools signified the most lucrative business in the Town of Brookhaven at the time. The current seal is a redesign, retaining the original elements, but adding the Town of Brookhaven and its 1655 date of settlement.

Early English settlers farmed, fished, and hunted whales. Brookhaven was largely agrarian, with each hamlet being limited to a handful of families yet containing miles of land. This economy was supplemented in coastal sections with fishing. A major commercial center did non exist in Brookhaven until the early 19th century.

Brookhaven was founded primarily by English colonists that partook in the settlement of Southold, New York, and was likewise under the jurisdiction of the theocratic New Haven Colony in the modern state of Connecticut. Brookhaven was transferred to the more secular Hartford-led Connecticut Colony in 1662. Following the English takeover of New Netherland in 1664, the new English colony of New York laid claim to Long Island and brought Brookhaven into its jurisdiction.

During the early stages of the American Revolutionary War, Brookhaven and the rest of Long Island were captured by British forces, and many residents sided with the British as loyalists to the English crown. Nevertheless, Brookhaven had multiple episodes of celebrated American activity during the war. This included the actions of the Culper Spy Ring, a spy network working for George Washington that largely consisted of Brookhaveners working in occupied territory. Another episode was Benjamin Tallmadge's successful raid from across Long Island to the British stronghold at the Manor St. George, wherein his raiding party rowed from Connecticut to Cedar Beach and marched across Long Island, culminating in the Battle of Fort St. George and burning of the defensive structure. A more minor skirmish occurred within the settlement of Setauket, where the British had repurposed the local Protestant church as a fortress. Gunshots were fired and some bullet holes remain within the walls of the adjacent Caroline Church.

In the mid-19th century, several communities in Brookhaven prospered as shipbuilding ports. The most successful of these are the villages of Port Jefferson and Patchogue, which remain the township's most bustling traditional downtowns. Whaling and cordwood industries also developed in Brookhaven.

Railroads reached Brookhaven in the mid-19th century, beginning in 1843 with trains reaching inland to Ronkonkoma. The earliest equivalent to the North Shore's present-day Port Jefferson Branch began operation in 1873. The Bridgeport & Port Jefferson Ferry has been operating between Port Jefferson and Bridgeport, Connecticut since 1888 and was partly founded by legendary circus-master P.T. Barnum.

From the end of the late 19th century until the middle of the 20th, many communities along the North Shore and South Shore became successful resort towns. These hosted many urban residents from nearby New York City, including numerous celebrities of the era, during the summer months. Meanwhile, many rural sections across Brookhaven served as campgrounds for youth clubs.

During the Gilded Age, Brookhaven underwent some of the same changes that affected Long Island's Gold Coast. Waterfront areas along the North Shore, including Belle Terre and Old Field, transformed from small fishing communities into fashionable and exclusive enclaves of the moneyed elite.

A variety of notable research occurred in Brookhaven around the turn of the 20th century. Inventor Nikola Tesla had a research center in Shoreham, which featured the large Wardenclyffe Tower. As of 2016 this site is being renovated as the Tesla Science Center at Wardenclyffe. Guglielmo Marconi opened one of the world's largest radio facilities, known as RCA's Radio Central, in Rocky Point.

In the post-war era, Brookhaven experienced a massive population boom. This was partly due to its proximity to New York City along the Long Island Rail Road and the highway system of Robert Moses. The township's most internationally renowned institutions, Stony Brook University and Brookhaven National Lab, both date to the years following World War II.

During the mid-century, a number of major transformations were conducted by philanthropist Ward Melville in the Three Village area (The Setaukets, Stony Brook, and Old Field). Melville, an Old Field resident and owner of what later became CVS Corporation, used his fortune to transform Northwestern Brookhaven to his vision of an idealized New England-style region. To this end, he ordered the construction in 1939 of the Stony Brook Village Center, a picturesque commercial center set on village green in Stony Brook, with clapboard buildings designed to look as if they had colonial origins. In 1962, Ward Melville donated 400 acres of land for the relocation of now-named Stony Brook University from Oyster Bay.

Brookhaven National Laboratory (BNL) sits on the site of Camp Upton, a United States army installation that was used as a training ground in World War I and World War II by thousands of soldiers including composer Irving Berlin. In 1946 ownership of the grounds was transferred for use by the new national laboratory, which began operating the following year. Brookhaven researchers have since made such diverse contributions as patenting Maglev, designing one of the first video games, detecting the first solar neutrinos, designing pollutant-eating bacteria, creating the first PET scan, and various contributions to particle physics. The laboratory contains the Relativistic Heavy Ion Collider (RHIC) and the National Synchrotron Light Source. Noted physicist and Nobel laureate I.I. Rabi was instrumental in the national laboratory's establishment.

As of the census of 2000, there were 448,248 people, 146,828 households, and 112,910 families residing in the town. The population density was 1,728.8 people per square mile (667.5/km²). There were 155,406 housing units at an average density of 599.4 per square mile (231.4/km²). The racial makeup of the town was 68.43% White, 24.33% Black or African American, 0.23% Native American, 2.90% Asian, 0.03% Pacific Islander, 2.21% from other races, and 1.87% from two or more races. Hispanic or Latino people of any race were 8.04% of the population.

There were 146,828 households, out of which 38.9% had children under the age of 18 living with them, 62.3% were married couples living together, 10.6% had a female householder with no husband present, and 23.1% were non-families. 17.9% of all households were made up of individuals, and 7.2% had someone living alone who was 65 years of age or older. The average household size was 2.97 and the average family size was 3.37.

In the town, the population was spread out, with 26.7% under the age of 18, 8.9% from 18 to 24, 31.5% from 25 to 44, 22.7% from 45 to 64, and 10.1% who were 65 years of age or older. The median age was 35 years. For every 100 females, there were 96.7 males. For every 100 females age 18 and over, there were 93.8 males.

According to a 2006 estimate, the median income for a household in the town was $73,566, and the median income for a family was $84,705. Males had a median income of $48,601 versus $32,157 for females. The per capita income for the town was $24,191. About 3.9% of families and 5.9% of the population were below the poverty line, including 6.5% of those under age 18 and 6.6% of those age 65 or over.