Despite the controversy, Reuters reported on Tuesday that two nuns had been spotted at the fast-food branch on Borgo Pio, a short distance away from St Peter's Basilica.
The restaurant, based in a building owned by the Holy See but not actually in the Vatican, is thought to be bringing a 30,000 Euro annual boost to the Vatican's coffers.
Speaking with the La Repubblica newspaper in October last year, Cardinal Elio Sgreccia branded allowing the McDonald's franchise to open "a perverse decision", claiming the space should be used to help the poor instead.
He told the Italian newspaper: "It would be better to use those spaces to help the needy of the area, spaces for hospitality, shelter and help for those who suffer, as the Holy Father teaches."
Some church leaders were said to be concerned the restaurant could spoil the area's "architectural traditions", while another cardinal called on Pope Francis to block the move.
Meanwhile, McDonald's said: "As is the case whenever McDonald's operates near historic sites anywhere in Italy, this restaurant has been fully adapted with respect to the historical environment."