The fire broke out in the intensive care unit of the Bambin Gesu' hospital, one of the largest children's clinics in Europe, on Friday.
No victims have been reported in the fire. Forty-eight people, who received severe smoke inhalation injuries while trying to escape from the burning building, were hospitalized, media reports said. Among them are several doctors who helped rescuers evacuate children from the hospital.
Fifteen firefighter units were involved in extinguishing the blaze. Their efforts were reportedly hampered by a large number of cars parked near the hospital building.
Rome Mayor Gianni Alemanno, who arrived at the site late on Friday, said the situation was "under control."
Vatican Secretary of State Cardinal Tarcisio Bertone also visited the hospital.
The causes of the fire are being investigated.