Challoner was educated at the English College at Douai, France, where he was ordained (1716) and appointed vice president and professor of theology (1720). In 1730 he was sent to London, where the Roman Catholic community was small and harassed, and in 1741 was consecrated titular bishop of Debra. In 1758 he became vicar apostolic of the London district, where he increased the number of converts to Catholicism, founded the Benevolent Society for the Relief of the Aged and Infirmed Poor, and strengthened Roman Catholics against the persecution prevalent in 18th-century England.

Challoner was the author of many books, including The Garden of the Soul (1740), a popular manual of prayer, and Meditations for Every Day of the Year (1753), frequently reprinted and used by non-Catholics. His carefully researched historical works include Britannia Sancta, an account of the lives of traditional British saints (1745), and British Martyrology (1761). Challoner's purpose in his revision of the Douai-Reims version (1749–50) was to make the Bible more readable by modernizing the language and to correct earlier errors.