The Museum occupies large part of the former Vallombrosan convent dedicated to San Salvi and derives its name from the grandiose fresco in the refectory of the Last Supper by Andrea del Sarto, one of the masterpieces of Florentine Renaissance painting. The spacious conventual rooms are hung with important early Cinquecento paintings which document the development of Florentine painting of the period. They include works by Pontormo, Andrea del Sarto, Franciabigio, Raffaellino del Garbo, Giuliano Bugiardini, Bacchiacca and others. Also exhibited here is the funerary monument of St Giovanni Gualberto, founder of the Vallombrosan Order, which was commissioned from Benedetto da Rovezzano in 1505 but remained unfinished.
A large study collection of paintings belonging to the museums in Florence is installed on the upper floor.