Over 12,000 people have joined a Facebook campaign to stop the fast food chain opening up in the historic area, and mayor Dario Nardella has also spoken out to condemn the plan.
The growing Facebook group has shared an edited photo of the iconic David statue depicting him as overweight - highlighting their view that the restaurant would threaten the city's cultural heritage - and invited supporters to gather in the square on Sunday for a 'flash mob'. Over 2,000 people have signed a petition which will be presented to the mayor.
The owners of the page said they wanted to make their point "peacefully and with irony, because we are Florentines".
The city's mayor, Nardella, wrote on Facebook: “To be clear, I am against the opening of a McDonald’s in the piazza del Duomo. It doesn’t agree with the battle which we have fought for years against fast food and mini markets for the protection of the city’s traditions and identity… We will do all that we can to avoid this.”
The fast food chain already has several restaurants in the city, with three others in the city centre just a short walk from the square, which is a Unesco World Heritage Site.
McDonald's - along with other foreign fast food chains - has had a problematic history in Italy. Its first restaurant opened up near the Spanish steps in Rome 30 years ago, sparking protests. Fashion brand Valentino, which has its Rome headquarters nearby, complained about the smells and noise from the restaurant, and the opening also sparked the now global Slow Food movement.
In 2013, the mayor of a Tuscan town complained about the use of images of its rolling hills in a McDonald's advert to promote a new burger. The recent arrival of Domino's and Starbucks in a country that takes pizza and coffee very seriously has also provoked controversy.
And 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.