Southerne was educated at Trinity College, Dublin, but spent his life after about 1680 in London, where he began to study law. His first play, The Loyal Brother, was produced at London's Drury Lane Theatre in 1682. From 1685 to 1688 he was soldiering, but he wrote several other plays and contributed prologues and epilogues to John Dryden's plays.

Both of Southerne's principal works were based on novels by Aphra Behn, a popular 17th-century novelist and poet. In their mingling of pathos with a sometimes flaccid rhetoric, they owed much to the 17th-century dramatist Thomas Otway, as well. The Fatal Marriage anticipated 18th-century domestic tragedy, and Oroonoko showed affiliations with the earlier heroic plays of Dryden. The role of Isabella, which was first played by the great English actress Elizabeth Barry, gave Sarah Siddons one of her major successes a century later. The character of Oroonoko, an African prince enslaved in the English colony of Surinam, marked one of the first literary appearances of the “noble savage,” and the play was a notably early English condemnation of the slave trade. As well as writing several other plays—lively comedies of manners and frigid tragedies in Roman settings—Southerne also revised and finished Dryden's tragedy Cleomenes (1692).