Although he was born in Westminster, London, Jonson claimed his family was of Scottish Border country descent, and this claim may have been supported by the fact that his coat of arms bears three spindles or rhombi, a device shared by a Borders family, the Johnstones of Annandale. His father died a month before Ben's birth, and his mother remarried two years later, to a master bricklayer. Jonson attended school in St. Martin's Lane, and was later sent to Westminster School, where one of his teachers was William Camden. Jonson remained friendly with Camden, whose broad scholarship evidently influenced his own style, until the latter's death in 1623. On leaving, Jonson was once thought to have gone on to the University of Cambridge; Jonson himself said that he did not go to university, but was put to a trade immediately: a legend recorded by Fuller indicates that he worked on a garden wall in Lincoln's Inn. He soon had enough of the trade, probably bricklaying, and spent some time in the Low Countries as a volunteer with the regiments of Francis Vere. In conversations with the poet William Drummond, subsequently published as the Hawthornden Manuscripts, Jonson reports that while in the Netherlands he killed an opponent in single combat and stripped him of his weapons.

Ben Jonson married, some time before 1594, a woman he described to Drummond as "a shrew, yet honest." His wife has not been definitively identified, but she is sometimes identified as the Ann Lewis who married a Benjamin Jonson at St Magnus-the-Martyr, near London Bridge. The registers of St. Martin's Church state that his eldest daughter Mary died in November, 1593, when she was only six months old. His eldest son Benjamin died of the plague ten years later (Jonson's epitaph to him On My First Sonne was written shortly after), and a second Benjamin died in 1635. For five years somewhere in this period, Jonson lived separately from his wife, enjoying instead the hospitality of Lord Aubigny.

By the summer of 1597, Jonson had a fixed engagement in the Admiral's Men, then performing under Philip Henslowe's management at The Rose. John Aubrey reports, on uncertain authority, that Jonson was not successful as an actor; whatever his skills as an actor, he was evidently more valuable to the company as a writer.

By this time, Jonson had begun to write original plays for the Lord Admiral's Men; in 1598, he was mentioned by Francis Meres in his Palladis Tamia as one of "the best for tragedy." None of his early tragedies survive, however. An undated comedy, The Case is Altered, may be his earliest surviving play.

In 1597, a play co-written with Thomas Nashe entitled The Isle of Dogs was suppressed after causing great offence. Arrest warrants for Jonson and Nashe were subsequently issued by Elizabeth's so-called interrogator, Richard Topcliffe. Jonson was jailed in Marshalsea Prison and famously charged with "Leude and mutynous behavior", while Nashe managed to escape to Great Yarmouth. A year later, Jonson was again briefly imprisoned, this time in Newgate Prison, for killing another man, an actor Gabriel Spenser, in a duel on 22 September 1598 in Hogsden Fields, (today part of Hoxton). Tried on a charge of manslaughter, Jonson pleaded guilty but was subsequently released by benefit of clergy, a legal ploy through which he gained leniency by reciting a brief bible verse (the neck-verse), forfeiting his 'goods and chattels' and being branded on his left thumb.

In 1598, Jonson produced his first great success, Every Man in his Humour, capitalising on the vogue for humour plays that had been begun by George Chapman with An Humorous Day's Mirth. William Shakespeare was among the first cast. This play was followed the next year by Every Man Out of His Humour, a pedantic attempt to imitate Aristophanes. It is not known whether this was a success on stage, but when published, it proved popular and went through several editions.

Jonson's other work for the theater in the last years of Elizabeth I's reign was unsurprisingly marked by fighting and controversy. Cynthia's Revels was produced by the Children of the Chapel Royal at Blackfriars Theatre in 1600. It satirized both John Marston, who Jonson believed had accused him of lustfulness, probably in Histrio-Mastix, and Thomas Dekker, against whom Jonson's animus is not known. Jonson attacked the same two poets again in 1601's Poetaster. Dekker responded with Satiromastix, subtitled "the untrussing of the humorous poet". The final scene of this play, whilst certainly not to be taken at face value as a portrait of Jonson, offers a caricature that is recognisable from Drummond's report - boasting about himself and condemning other poets, criticising performances of his plays, and calling attention to himself in any available way.

This "War of the Theatres" appears to have been concluded with reconciliation on all sides. Jonson collaborated with Dekker on a pageant welcoming James I to England in 1603 although Drummond reports that Jonson called Dekker a rogue. Marston dedicated The Malcontent to Jonson and the two collaborated with Chapman on Eastward Ho, a 1605 play whose anti-Scottish sentiment landed both authors in jail for a brief time.

At the beginning of the reign of James I of England in 1603 Jonson joined other poets and playwrights in welcoming the reign of the new king. Jonson quickly adapted himself to the additional demand for masques and entertainments introduced with the new reign and fostered by both the king and his consort Anne of Denmark.

Jonson flourished as a dramatist during the first decade or so of James's reign; by 1616, he had produced all the plays on which his reputation as a dramatist depends. These include the tragedy of Catiline (acted and printed 1611), which achieved only limited success, and the comedies Volpone, (acted 1605 and printed in 1607), Epicoene, or the Silent Woman (1609), The Alchemist (1610), Bartholomew Fair (1614) and The Devil is an Ass (1616). The Alchemist and Volpone appear to have been successful at once. Of Epicoene, Jonson told Drummond of a satirical verse which reported that the play's subtitle was appropriate, since its audience had refused to applaud the play (i.e., remained silent). Yet Epicoene, along with Bartholomew Fair and (to a lesser extent) The Devil is an Ass have in modern times achieved a certain degree of recognition. While his life during this period was apparently more settled than it had been in the 1590s, his financial security was still not assured. In 1603, Overbury reported that Jonson was living on Aurelian Townsend and "scorning the world."