The food giant wants to open a new store in Piazza del Duomo near to where the UNESCO World Heritage site building is housed.
Despite the opposition which now has the backing of the Mayor of Florence, the US firm says it has an agreement with his office.
"There's nothing ideological about it, but we are against the opening of a McDonald's near the Duomo and in other historic piazzas. We feel that the historic environment of the city needs to be safeguarded," the mayor said.
New rules in the city mean new businesses have to source their products from Tuscany to preserve local traditions and it's claimed McDonalds was happy to abide.
"For months we have been talking to the mayor and the council, who even complimented us on our plans. Why these insults now? Politics seems to lie behind it," the head of McDonald's in Italy, Roberto Masi told La Repubblica.
Cathedral authorities are yet to comment on the potential new store.