The chapel of San Ambrosio is the first of the chapels attached to the western wall. It is one of the oldest chapels in the building and has a wooden ceiling with only a few remains of the original coffered ceiling.

It is not possible to determine the name of the person who founded the chapel, which was renovated by the master builder Juan Ruíz de Córdoba in 1528. In 1839 it was restored by Pedro María Villavicencio, who was also a master of the cathedral. The persistence of the cathedral dignity of maestrescuelas related to this chapel is due to the fact that the enclosure and the dedication to Saint Ambrose were linked to the patronage of the maestrescolía.

The altarpiece, which was commissioned from the Sánchez de Rueda brothers in 1717, consists of a bench, body and semicircular attic, and the bench, by means of four bases, serves as a support for the supports of the main body. It is articulated from Solomonic columns, and is decorated with paintings of the Ascension and the Epiphany, of poor quality, executed by Pedro Ruiz Morián, and its execution having been arranged in 1723. The central street of the altarpiece, which is twice as wide as the side streets, is occupied by an image of Saint Ambrose from the 17th century. In the attic of the altarpiece there is a carved Calvary from the end of the 16th century.