The devil was hidden in the details of clouds at the top of fresco number 20 in the cycle of the scenes in the life and death of St Francis painted by Giotto in the 13th century.
The discovery was made by Italian art historian Chiara Frugone. It shows a profile of a figure with a hooked nose, a sly smile, and dark horns hidden among the clouds in the panel of the scene depicting the death of St Francis.
The figure is difficult to see from the floor of the basilica but emerges clearly in close-up photography.
Sergio Fusetti, the chief restorer of the basilica, said Giotto probably never wanted the image of the devil to be a main part of the fresco and may have painted it in among the clouds "to have a bit of fun."
The master may have painted it to spite someone he knew by portraying him as a devil in the painting, Fusetti said on the convent's website.
The artwork in the basilica in the convent where St Francis is buried was last restored after it was severely damaged by an earthquake in 1997.
(Reporting By Philip Pullella; Editing by Sophie Hares)