Born in Gubbio, he succeeded his father Federico da Montefeltro as Duke of Urbino in 1482.

In 1496, while fighting for the pope near Bracciano, Guidobaldo was taken prisoner by the Orsini and the Vitelli, being freed the following year.

He adopted as his heir Francesco Maria della Rovere, his sister's child and nephew of Pope Julius II, thus uniting the seigniory of Senigallia with Urbino.

Suffering from gout, Guidobaldo died in Fossombrone at the age of 36, and was succeeded by his nephew.

In 2023, Guidobaldo gained interest on social media when American sports fans noted how he appeared striking similar to NFL quarterback Trevor Lawrence of the Jacksonville Jaguars.