The September 1590 papal conclave, attended by 54 cardinals, elected Cardinal Giovanni Castagna as Pope Urban VII.

The conclave lasted a week, and was heavily swayed by the influence of the twenty-two Spanish cardinals. Castagna, who had been favored by Sixtus V as a successor and was on the list of candidates whom Philip II of Spain had approved, was a favorite from the beginning, although a significant opposing faction supported Marco Antonio Colonna. Castagna's poor health, which would ultimately kill him after a papacy of only thirteen days, made him a more palatable candidate to cardinals who were outraged at Philip's attempts to sway the election.

Contemporary newspapers reported that a disagreement between Cardinals Ascanio Colonna and Francesco Sforza di Santa Fiora during the conclave nearly became violent.