Bentley was educated at Wakefield Grammar School and St. John's College, Cambridge. In 1689 he made the acquaintance of John Mill at Oxford, who asked him to look through the proof sheets of the Oxford edition of the chronicler John Malalas. The request gave rise to Bentley's Epistola ad Joannem Millium (1691), a short treatise in which his skill in textual emendation and his knowledge of ancient metre were strikingly displayed.

Bentley was appointed Boyle lecturer at the University of Oxford in 1692, and in 1694 he became keeper of the Royal Library and fellow of the Royal Society. In 1699 he published his Dissertation upon the Epistles of Phalaris, a work in which he attacked the authenticity of the epistles, bringing all his learning and critical powers to bear in proof of their spuriousness.

In 1700 Bentley was chosen master of Trinity College, Cambridge, and in 1717 he became regius professor of divinity. His tenure as master was marked by friction and litigation. His domineering temper and his contemptuous treatment of the fellows led to various attempts to secure his ejection and embroiled him in controversy and feuding for the next 30 years.

Through it all, however, Bentley continued his classical studies. He published a critical appendix to John Davies' edition of Cicero's Tusculan Disputations in 1709, and two years later he published an edition of Horace. His later works include an edition of Terence, published in 1726, together with the fables of Aesop and the Sententiae of Publilius Syrus, and in 1739 an edition of Marcus Manilius. On other classical authors, such as Nicander, Plautus, Lucretius, and Lucan, he left notes, which were published after his death. Bentley made a particularly important scholarly contribution through his discovery that a sound (represented in transcriptions of some Greek dialects by the digamma, a letter not used in the modern Greek alphabet) was present in certain Homeric Greek words, though not represented by any letter when the words were written.