The Cenacolo dates to the early 16th century when it was the refectory of the Vallombrosan abbey of San Salvi. It was frescoed by Andrea del Sarto between 1511 and 1527, and was used by the enclosed nuns of Beata Umiltà until the early 19th century.

The refectory contains the outstanding fresco by Andrea del Sarto depicting the Last Supper, considered the painter's masterpiece and carried out around 1520.

The Cenacolo also houses a collection of panels and altarpieces from the Florentine school, many of which originated from suppressed Florentine churches and monasteries.

Also by Andrea del Sarto the Annunciation, Pietà and Noli me tangere. Works by contemporary artists or followers of Andrea del Sarto are the Madonna and Child by Giuliano Bugiardini, the San Zanobi Reviving a Child and the Translation of the body of San Zanobi by Ridolfo del Ghirlandaio, the Adoration of the Shepherds, Sacred Conversation and Noli me tangere by Franciabigio, the Madonna and Child with the Young St John by Pontormo.