The 37-year-old Canadian man had climbed the 463 steps to the top of the cathedral dome when he had the urge to urinate, La Nazione reported.
Realizing that architect Filippo Brunelleschi had not had the foresight to include toilets in his 15th century dome design, the tourist decided to pee in the porter's lodge.
He was spotted by security staff who called on city police to intercept the Canadian when he returned to the square below. Officers handed out a €400 fine to the tourist, who reportedly tried to justify his actions by complaining of the lack of bathroom facilities.
Brunelleschi's dome broke architectural boundaries when it was constructed between 1418 and 1434. Described by the Museum of Florence as “a masterpiece capable of withstanding lightning, earthquakes and the passage of time,” the dome is famous for being built without scaffolding to hold it together.
It was instead created by building two domes, an outer one and an inner frescoed structure, with a space between which visitors now clamber up. The small rooftop affords impressive views of Florence, but there is no space for bathroom facilities.
Millions of tourists visit the Renaissance city each year, a few of which find themselves in trouble for misbehaving.
In March a Japanese woman was fined for scrawling her name on Brunelleschi's dome with eyeliner, while last year an American woman was spotted urinating in a nearby square.
Italians have also been caught rule-breaking, with one man in September stopped by police for urinating on the wall of Florence's Santa Croce church.