The deuterocanonical Book of Judith tells how Judith served her people by seducing and pleasuring Holofernes, the Syrian General. Judith gets Holofernes drunk, then seizes her sword and slays him: ‘Approaching to his bed, she took hold of the hair of his head’ (Judith 13:7–8).

Caravaggio's approach was, typically, to choose the moment of greatest dramatic impact: the moment of decapitation itself. The figures are set out in a shallow stage, theatrically lit from the side, isolated against the inky black background. Judith's maid Abra stands beside her mistress to the right as Judith extends her arm to hold a blade against Holofernes's neck; lying on his stomach, neck contorted as he turns his head towards his assassin, he is vulnerable. X-rays have revealed that Caravaggio adjusted the placement of Holofernes' head as he proceeded, separating it slightly from the torso and moving it minutely to the right. The faces of the three characters demonstrate the artist's mastery of emotion, Judith's countenance in particular showing a mix of determination and repulsion. Artemisia Gentileschi and others were deeply influenced by this work; while they even surpassed Caravaggio's physical realism, it has been argued that none matched his capture of Judith's psychological ambivalence.

The model for Judith is probably the Roman courtesan Fillide Melandroni, who posed for several other works by Caravaggio around this year; the scene itself, especially the details of blood and decapitation, were presumably drawn from his observations of the public execution of Beatrice Cenci in 1599.