The Basilica of San Petronio, dedicated to the city's patron saint, is located in Piazza Maggiore and is the largest and most important church in Bologna.

Its construction began in 1390 under the supervision of Antonio di Vincenzo. In 1514, Arduino degli Arriguzzi suggested a new Latin-cross model that would have been larger than St. Peter's Basilica in Rome but, according to legend, Pope Pius IV blocked its construction and insisted on the erection of the Archiginnasio instead. The façade also remained unfinished, being only partly covered in marble by Giacomo Ranuzzi starting from 1538. The interior, completed in different eras, is divided into three naves, which open onto twenty-two chapels decorated by artists, including Francesco Francia, Lorenzo Costa, Parmigianino and Donato Credi.

Famous is the Music Chapel of San Petronio, the oldest musical institution in Bologna (1436), whose most prestigious symbol is a still functioning organ, which, built around 1470 by Lorenzo da Prato, is the oldest in the world still in use. The Chapel of the Three Wise Men (IV) or Bolognini,  is also very popular among visitors. Its walls were sumptuously frescoed by Giovanni da Modena with scenes representing “Heaven” and “Hell”.

The sundial by the astronomer Cassini is also well-known and is the longest in the world, measuring 67.27 metres.