Son of a Protestant refugee of Tuscan origin, Florio studied at Oxford. From 1604 to 1619 Florio was groom of the privy chamber to Queen Anne.

In 1580 he translated, as Navigations and Discoveries (1580), Giovanni Battista Ramusio's account of the voyages of Jacques Cartier. Florio His Firste Fruites (1578), a grammar and a series of dialogues in Italian and English, was followed in 1591 by Florio's Second Frutes and by Giardino di ricreatione, a collection of more than 6,000 proverbs in Italian. His Italian-English dictionary, A Worlde of Wordes (1598), for which he drew heavily upon the works of Giordano Bruno, contains about 46,000 definitions. The second edition, Queen Anna's New World of Words (1611), was greatly enlarged.

In 1603 Florio produced his major translation, the Essais of Michel de Montaigne, which he revised in 1613. The freedom of this version is questionable by modern standards of accuracy, and the style is elaborate where Montaigne is subtle and terse, but the book is nevertheless thoroughly good reading.