An Italian art historian, Chiara Frugoni, spotted the devil's profile in the fresco's clouds, high up in the Basilica of St Francis in Assisi.
The face, with hooked nose and an evil smirk, is hard to see from the ground.
The fresco, featuring the death of St Francis, dates from the 13th Century. Giotto di Bondone is seen as one of the finest Early Renaissance artists.
Ms Frugoni said it was previously thought the first artist to conceal a portrait in clouds was Andrea Mantegna, in the 15th Century.