Gentileschi first studied with his half brother Aurelio Lomi. At some time in the late 1570s or early 1580s he went to Rome, where, with the landscape painter Agostino Tassi, he painted frescoes in churches of Santa Maria Maggiore, San Giovanni Laterano, and Santa Nicola in Carcere from about 1590 to 1600, executing figures for Tassi's landscapes.

In the first years of the 17th century Gentileschi came under the influence of Caravaggio, also in Rome at the time. His paintings of this period—e.g., David and Goliath (1610?) and St. Cecilia and the Angel (1610?)—employ Caravaggio's use of dramatic, unconventional gesture and monumental composition, his uncompromising realism and contemporary representation of figure types, and to some extent his strong chiaroscuro, or light-and-dark contrast.

Shortly afterward Gentileschi developed a Tuscan lyricism foreign to Caravaggio's almost brutal vitality, a lighter palette, and a more precise treatment reminiscent of his Mannerist (Mannerism) beginnings. From 1621 to 1623 Gentileschi was in Genoa, where he painted his masterpiece, The Annunciation (Annunciation) (1623), a work of consummate grace that shows a weakening of Caravaggio's influence. The composition still depends on dramatic gestures, here of the Virgin and the angel, and there is still a strong immediacy to the incident and an absence of idealization. The mood, however, is more restrained and lyrical than in his earlier works, the colours are light, and the earlier chiaroscuro is absent.

After a stay in France, Gentileschi traveled to England in 1626 at the invitation of King Charles I (Charles I). He remained there as court painter for the rest of his life, his work becoming increasingly conventional and decorative. His last major work is an ambitious series of ceiling paintings, which he worked on with his daughter, Artemisia, for the Queen's House in Greenwich; painted in 1638, the ceiling panels have since been removed to Marlborough House, London.