By 1481 Pinturicchio was associated with the Umbrian artist Perugino, whose influence on him was to be permanent. It is generally agreed that he assisted Perugino on some of the frescoes (“Journey of Moses” and the “Baptism of Christ”) in the Sistine Chapel in the Vatican (1481/82). In the 1480s he worked in the Bufalini Chapel in Santa Maria in Aracoeli and in Santa Maria del Popolo (both in Rome).

Pinturicchio's most important work of this period was the decoration of the suite of six rooms in the Vatican (Vatican palace) known as the Borgia Apartments for Pope Alexander VI between 1492 and 1494. In these frescoes he retains Perugino's figure types but lacks his clarity of conception. Instead, Pinturicchio relies on brilliant, often jarring colours, gilding, and ancient Roman ornamental motifs.

Pinturicchio's last major works were the 10 scenes from the life of Pope Pius II painted (1503–08) in fresco in the Piccolomini Library in Siena. In these, space, colour, and detail are handled with a crisp proficiency that may have influenced Raphael.