The beheading of Holofernes by Judith is recounted 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 Jewish widow, enters the tent of Assyrian general Holofernes under the guise of seduction, as he was preparing to destroy Judith's home, the city of Bethulia. He passes out intoxicated and is decapitated by Judith; his head is taken away in a basket (often depicted as being carried away by an elderly female servant).

Artists have mainly chosen one of two possible scenes (with or without the servant): the decapitation, with Holofernes supine on the bed, or Judith the heroine holding or carrying the head.

In European art, Judith is very often accompanied by her maid at her shoulder, which serves to distinguish her from Salome, who also carries her victim's head on a silver charger (plate). However, a northern tradition developed whereby Judith had both a maid and a charger, taken by Erwin Panofsky as an example of the knowledge needed in the study of iconography. For many artists and scholars, Judith's sexualized femininity is sometimes contradictorily combined with more stereotypical masculine aggression. Judith was one of the virtuous women whom Van Beverwijck mentioned in his published apology (1639) for the superiority of women to men, and a common example of the Power of Women iconographic theme in the Northern Renaissance.

The Book of Judith in the Bible was accepted by Jerome as canonical and accepted in the Vulgate and was referred to by Clement of Rome in the late first century (1 Clement 55), and thus images of Judith were as acceptable as those of other scriptural women. In early Christianity, however, images of Judith were far from sexual or violent: she was usually depicted as "a type of the praying Virgin or the church or as a figure who tramples Satan and harrows Hell," that is, in a way that betrayed no sexual ambivalence: "the figure of Judith herself remained unmoved and unreal, separated from real sexual images and thus protected."

Judith and Holofernes, the famous bronze sculpture by Donatello, bears the implied allegorical subtext that was inescapable in Early Renaissance Florence, that of the courage of the commune against tyranny.

In the late Renaissance, Judith changed considerably, a change described as a "fall from grace"—from an image of Mary she turns into a figure of Eve. Early Renaissance images of Judith tend to depict her as fully dressed and desexualized; besides Donatello's sculpture, this is the Judith seen in Sandro Botticelli's The Return of Judith to Bethulia (1470–1472), Andrea Mantegna's Judith and Holofernes (1495, with a detached head), and in the corner of Michelangelo's Sistine Chapel (1508–1512). Later Renaissance artists, notably Lucas Cranach the Elder, who with his workshop painted at least eight Judiths, showed a more sexualized Judith, a "seducer-assassin": "the very clothes that had been introduced into the iconography to stress her chastity become sexually charged as she exposes the gory head to the shocked but fascinated viewer", in the words of art critic Jonathan Jones. This transition, from a desexualized image of Virtue to a more sexual and aggressive woman, is signaled in Giorgione's Judith (c. 1505): "Giorgione shows the heroic instance, the triumph of victory by Judith stepping on Holofernes's severed, decaying head. But the emblem of Virtue is flawed, for the one bare leg appearing through a special slit in the dress evokes eroticism, indicates ambiguity and is thus a first allusion to Judith's future reversals from Mary to Eve, from warrior to femme fatale." Other Italian painters of the Renaissance who painted the theme include Botticelli, Titian, and Paolo Veronese.

Especially in Germany an interest developed in female "worthies" and heroines, to match the traditional male sets. Subjects combining sex and violence were also popular with collectors. Like Lucretia, Judith was the subject of a disproportionate number of old master prints, sometimes shown nude. Barthel Beham engraved three compositions of the subject, and other of the "Little Masters" did several more. Jacopo de' Barberi, Girolamo Mocetto (after a design by Andrea Mantegna), and Parmigianino also made prints of the subject.

Judith remained popular in the Baroque period, but around 1600, images of Judith began to take on a more violent character, "and Judith became a threatening character to artist and viewer." Italian painters including Caravaggio, Leonello Spada, and Bartolomeo Manfredi depicted Judith and Holofernes; and in the north, Rembrandt, Peter Paul Rubens, and Eglon van der Neer used the story. The influential composition by Cristofano Allori (c. 1613 onwards), which exists in several versions, copied a conceit of Caravaggio's recent David with the Head of Goliath: Holofernes' head is a portrait of the artist, Judith is his ex-mistress, and the maid her mother. In Artemisia Gentileschi's painting Judith Slaying Holofernes (Naples), she demonstrates her knowledge of the Caravaggio Judith Slaying Holofernes of 1612; like Caravaggio, she chooses to show the actual moment of the killing. A different composition in the Pitti Palace in Florence shows a more traditional scene with the head in a basket.