By 1600 Middleton had spent two years at Oxford and had published three books of verse. He learned to write plays by collaborating with Thomas Dekker (Dekker, Thomas), John Webster (Webster, John), and others for the producer Philip Henslowe (Henslowe, Philip).

A popular playwright, he was often commissioned to write and produce lord mayor's pageants and other civic entertainments, and in 1620 he was appointed city chronologer. His chief stage success was A Game at Chess (1625), in which the Black King and his men, representing Spain and the Jesuits, are checkmated by the White Knight, Prince Charles. This political satire drew crowds to the Globe Theatre until the Spanish ambassador protested and James I suppressed the play.

Middleton's masterpieces are two tragedies, Women Beware Women (1621?, published 1657) and The Changeling (1622, with William Rowley (Rowley, William); published 1653). His comedies picture a society dazzled by money in which most people grasp for all they can get, by any means. Michaelmas Term (1605?, published 1607) is one of the richest in irony. In A Trick to Catch the Old One (1606?, published 1608) two rival usurers are so eager to score over each other that both are taken in by a clever nephew. A Trick was entered for licensing with an unattributed play entitled The Revenger's Tragedy (1607). Modern scholarship attributes the latter to Middleton, although Cyril Tourneur (Tourneur, Cyril) is sometimes given as the author. In A Mad World, My Masters (1604?, published 1608) an old country gentleman prides himself on his generosity to all except his grandson and heir.

The Roaring Girl (1604–10?, with Dekker; published 1611) depicts events in the life of the notorious criminal Moll Frith ( Moll Cutpurse), who dressed as a man and preferred her freedom to marriage. A Chaste Maid in Cheapside (1613?, published 1630) is an exuberant comedy that makes fun of naive or complacent London citizens.

Middleton's tragicomedies are farfetched in plot but strong in dramatic situations. A Fair Quarrel (1616?, with Rowley, published 1617) contains one of Middleton's few heroes, Captain Ager, with his conflicts of conscience. Most of Middleton's other plays are comedies. He collaborated with Dekker in The Honest Whore (1604), and with Rowley and Philip Massinger (Massinger, Philip) in The Old Law (1618?, published 1656). In 2007 all the works attributed to Middleton were published together, for the first time, as Thomas Middleton: The Collected Works (eds. Gary Taylor and John Lavagnino).