The scene of the Last Supper, a subject widely used in refectories (the dining halls where monks and nuns ate their meals), occupies the lower section of a fresco that originally covered the entire west wall of the room. Above it were three additional scenes from the life of Christ: the Resurrection, the Crucifixion, and the Deposition, which were later detached from the wall for conservation purposes. Characterized by geometric rigor and precise use of perspective, the scene takes place in a richly decorated interior, where numerous carefully rendered details can be admired: draperies, ornamental friezes, and fine marble panels; the lighting, coming from two windows located on the right, originally mirrored the natural light in the room (whereas today the natural light comes from the left), while the figures are characterized by a wide variety of gestures and postures, which contribute to the realism of the scene. Although Castagno's work would later serve as a model for Leonardo da Vinci's Last Supper, it still follows the traditional iconography of the subject: Judas is seated on the opposite side of the table from the other figures (whereas in Leonardo's version, Christ and all the Apostles are depicted on the same side of the table); other two details contribute to emphasizing Judas's marginalization and his role as the traitor: his appearance, which draws on the iconography of satyrs, and the fact that at the bottom of the scene there is a low step bearing the names of all the Apostles, except for his.

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