Judith Beheading Holofernes is a painting of the biblical episode by Caravaggio, painted in c. 1598–1599 or 1602, in which the widow Judith stayed with the Assyrian general Holofernes in his tent after a banquet then decapitated him after he passed out drunk. The painting was rediscovered in 1950 and is part of the collection of the Galleria Nazionale d'Arte Antica in Rome. The exhibition 'Dentro Caravaggio' Palazzo Reale, Milan, suggests a date of 1602 on account of the use of light underlying sketches not seen in Caravaggio's early work but characteristic of his later works. The exhibition catalogue also cites biographer artist Giovanni Baglione's account that the work was commissioned by Genoa banker Ottavio Costa.

The account of the beheading of Holofernes by Judith is given in the deuterocanonical Book of Judith, and is the subject of many paintings and sculptures from the Renaissance and Baroque periods. In the story, Judith, a beautiful widow, is able to enter the tent of Holofernes because of his desire for her. Holofernes was an Assyrian general who was about to destroy Judith's home, the city of Bethulia. Overcome with drink, he passes out and is decapitated by Judith; his head is taken away in a basket.