William Randolph was baptized in Moreton Morrell, Warwickshire, England on 7 November 1650, He was the son of Richard Randolph (21 February 1621 – 2 May 1678) and Elizabeth Ryland (1625–ca. 1669). Richard Randolph was originally from Houghton Parva, a small village east of Northampton, where his father was a "steward and servant" to Edward la Zouche, 11th Baron Zouche (1556–1625), having previously served in that same capacity to Sir George Goring, a landowner in Sussex. His mother was a daughter of John Ryland of Warwick. william was the second of seven Randolph children, all born in Moreton Morrell between 1647 and 1657.

Although his father's older half-brother, the poet Thomas Randolph, attended Westminster School and Cambridge University, he did so largely on scholarship and there is no record of any other members of William's family having attended either public school or university. At some point in the late 1650s or 1660s, his parents moved to Dublin, where they both died, his mother around 1669 and his father in 1671, so william may well have spent the bulk of his formative years in Ireland. It is also known that William's uncle, Henry Randolph (1623-?), in 1669 traveled to Britain from Virginia, to which place he had emigrated around 1642. Henry probably encouraged his nephew at that time to return with him to the Chesapeake. In any case, william Randolph was in the colony by 12 February 1672 when he appears in the record as witness to a land transaction.

Randolph held multiple official appointments. At the local level, he became clerk of Henrico County Court in 1673 and held the position until he was asked to serve as a justice of the peace in 1683. He also served as sheriff and coroner.

The Chesapeake economy was centered around tobacco, grown within the English mercantile system for export to markets in Britain and Europe. Randolph appears to have arrived in the province with little capital and few transatlantic connections. One Historian suggests that he started off in the colony as an "undertaker" building houses, but there is no evidence for it. By 1674 he had acquired enough money to import 12 persons into the colony and thereby earned his first of many land patents (between 1674 and 1697 he imported 72 servants and 69 slaves for which he collected patents for more than 7000 acres). In later years Randolph became a merchant and a planter, and co-owned several ships used to transport tobacco to England and goods back to Virginia. He established several of his sons as merchants and ship captains.

William Randolph owned a considerable number of slaves. This reflected the rise of slavery during his Business career. Around 1675 Governor Berkeley reported the population of the colony as 40,000, with 4,000 indentured servants and 2,000 slaves. But as the supply of indentured servants declined late in the 17th century, the planters turned to slaves for work in the labor-intensive tobacco cultivation.

In 1676 a Virginia colonist, Nathaniel Bacon, rebelled unsuccessfully against the colonial government and his estate was forfeited. This was Curles, located near Turkey Island. Randolph made an assessment of the property for Governor Berkeley and was allowed to buy it for his estimated price, adding 1,230 acres (5.0 km) to Randolph's previous land holdings. This conflict of interest was criticized by his neighbors.

In 1678 Mary Isham's brother died, making her the heiress to her father's large estate. william Randolph had married her before her brother's death, because the brother's will refers to her as "Mary Randolph".

Randolph represented Henrico County in every assembly of the House of Burgesses from 1684 to 1698, was Speaker of the House of Burgesses in 1698, and was the Clerk of the House from 1699 to 1702. He fell ill in August 1702 and his son, william, took his place. Randolph resigned the clerkship completely in March 1703.

Around 1700, when Randolph's political career was at its peak, he received land grants to almost 10,000 acres (40 km) of newly opened land near Richmond; a 3,256-acre (13.18 km) tract at Tuckahoe Creek and a 5,142-acre (20.81 km) plot at Westham. This land became the basis of the Tuckahoe and Dungeness Plantations, which were later founded by two of william Randolph's sons.

It is difficult to determine the acreage or number of slaves he owned at his death. His will has been transcribed and a copy appears on the internet, but portions are missing. One estimate is that he had 20,000 acres (81 km). He paid property taxes on 1,655 acres (6.70 km) in Surry County and 19,465 acres (78.77 km) in Henrico County in 1704.

Around 1675 he married Mary Isham (1660 Bermuda Hundred, James River, Henrico County, Virginia–25 December 1735 Turkey Island, Henrico County, Virginia), whose father, Henry Isham (c. 1628 Pytchley, Northamptonshire–c. 1676 Bermuda Hundred, James River, Henrico County, Virginia), was from a gentry family in Northamptonshire. After arriving in Virginia, Henry had married in 1659, a wealthy widow, Katherina Banks Royall (c. 1630 Canterbury, Kent–aft. 1 December 1686 Henrico County, Virginia).

The name "Bremo" comes from a Germanic word meaning "edge", in this case the edge of a river. The root also occurs in the English word "brim". The plantation identified with Edward Randolph was near Turkey Island, in Henrico County. The extant Bremo Plantation was established in the early 19th century in Fluvanna County, far to the west. These plantations are shown on the 1751 Fry-Jefferson map.

Most of the Randolphs, like the rest of the Virginia gentry, strongly supported the Revolution. However, John Randolph (son of Sir John), in opposition to both his brother Peyton and son Edmund, remained loyal to Great Britain and left Virginia. It was the period of william Randolph's most famous descendants. Thomas Jefferson wrote the Declaration of Independence, and 18-year-old John Marshall was at Valley Forge for the trying winter of 1777–1778.

In their wealth and social status, the Randolphs were much like other families of the Chesapeake elite. If anything set them apart it was their participation in the political life of the colony, clearly traceable to william Randolph's Example. Randolphs and close relatives formed the predominant political faction in the colonial government during the 18th century, with many members of the elected House of Burgesses and the appointed, and more exclusive, Council.

Randolph was a friend of william Byrd, and he served as an advisor to Byrd’s sons during their political careers. He is mentioned in one of Byrd's diaries as "Colonel Randolph", his militia title.