With over 40,000 items spanning a vast collection of painting, sculpture, goldware, furniture, textiles, ceramics, and prints, MNAA is one of the most visited museums in Portugal.

The museum's collection spans more than a millennium of art from Europe, Asia, Africa, and the Americas and includes notable artworks by Hieronymus Bosch, Raphael, Hans Holbein the Elder, Francisco de Zurbarán, Albrecht Dürer, Domingos Sequeira, and Giambattista Tiepolo, among numerous others.

Although primarily dedicated to nurturing new artists, that same year, the Academia founded the Galeria Nacional de Pintura ("National Gallery of Painting") on the lower levels of the same building, as a subsidiary division to select, care for and display some of the better pieces of the expropriated monastic art then in government storage.

[3] In the chaos and aftermath of the Portuguese Liberal Wars, some of the private art collections of ruined noble families were expropriated or found their way on to the market.

[3] On the initiative of Francisco de Sousa Holstein (son of the 1st Duke of Palmela, a vice-inspector of the Academia Real, in 1868, the Galeria's rooms were remodelled into a better exhibition space for its expanding collection and opened to the general public.

[3] Nonetheless, the facilities remained inadequate - it was terribly humid, cramped and still traversed by unrelated visitors to the Academia (which had, by now, become something of a social hang-out for dissheveled bohemian artists and students).

The collection includes painting, sculpture, metalwork, textiles, furniture, drawings, and other decorative art forms from the Middle Ages to the early 19th century.

The European painting section of the museum is significant and is represented by Jacob Adriaensz Backer, Bartolomé Bermejo, Triptych of the Temptation of St. Anthony by Hieronymus Bosch, Pieter Brueghel the Younger, David Gerard, Saint Jerome in His Study by Albrecht Dürer, Lucas Cranach, Piero della Francesca, Jan Gossaert, Hans Holbein the Elder, Pieter de Hooch, Adriaen Isenbrandt, Quentin Metsys, Hans Memling, Antonis Mor, Joachim Patinir, Jan Provost, Raphael, José Ribera, Andrea del Sarto, David Teniers the Younger, Tintoretto, Anthony van Dyck, Diego Velázquez, David Vinckeboons, Hendrick Cornelisz Vroom, Francisco de Zurbarán, François Boucher, Nicolas Poussin and others.

Perhaps the most famous work in the museum is the Saint Vincent Panels, which date from before 1470 and are attributed to Nuno Gonçalves, court painter of King Afonso V. Though the history of the polyptych remains enshrouded in mystery, the six large panels have been argued to portray people from all levels of late medieval Portuguese society venerating Saint Vincent, in what would be one of the first group portraits in European art.