The US fast-food chain told AFP Monday it was claiming 17.8 million euros ($19.65 million) in damages after the city rejected an application to open an outlet in the historic Piazza del Duomo, one of the most visited places in Europe.
McDonald's said the suit was being filed with the administrative court, which arbitrates in governance disputes in Italy. The firm gave no other details.
Florence's centre-left mayor, Dario Nardella, turned down McDonald's application in June, in a decision that was confirmed the following month by a technical panel in charge of preserving the city's ancient heart.
"McDonald's has the right to submit an application, because this is permitted under the law, but we also have the right to say no," Nardella told the city council, saying he wanted to support "traditional business" in the area.
"We don't have any prejudice" against McDonald's, Nardella said, noting that the company had opened restaurants "in other parts of town."
There are three other McDonald's restaurants in the city, just a short walk from the square - but opening in Piazza del Duomo itself, which is a Unesco World Heritage site, was a step too far for locals.
A Facebook campaign opposed to the restaurant received thousands of likes within hours, while residents also presented a petition to Nardella's office.
Campaigners shared images included an edited, overweight version of the famous statue of David by Michelangelo to highlight the effect a McDonald's restaurant would have on the city's cultural heritage.
According to several reports in the Italian media, McDonald's is particularly aggrieved in the case of Florence as it had promised to make major changes to its business model to fit in with local regulations.
Florence has taken the struggle to protect its culinary history particularly strongly. In March, the city passed a law aimed at ensuring that at least 70 percent of produce in all new eateries was locally sourced, amid worries that a growing number of cheap kebab shops and other fast food outlets aimed at tourists meant the city was at risk of losing its character.
Cardinals in a tizz
Worried about the proliferation of fast-food outlets catering to backpackers in the city of Michelangelo, Florence introduced new licensing regulations in January requiring restaurants in the historic heart to use "typical products" either of the city or from the Tuscany region.
The Piazza del Duomo - "Cathedral Square" - is a treasure of Gothic and Renaissance architecture. Its buildings include the domed Santa Maria del Fiore cathedral, the Giotto bell tower and the St. John Baptistery.
Last month, plans to open a McDonald's in a piazza next to Saint Peter's Square in Rome angered cardinals who live above the proposed site.
The scheme is "by no means respectful of the architectural traditions of one of the most characteristic squares which look onto the colonnade of Saint Peters," it quoted Cardinal Elio Sgreccia as saying.
In their expansion into historic locations in Europe, McDonald's and other fast-food chains often encounter opposition on cultural or architectural grounds.
Many disputes, though, end in a compromise, in such areas as menu choice, litter control and the design of the business facade.