They used what they said was washable yellow and red paint, before putting up a banner saying: "End animals in circuses."
Tourists in the historic city centre square took photos with their phones before cleaners arrived to wash off the paint, some of which dripped into the water.
In a statement, the group said the action marked the launch of the "Kimba campaign" to end the "violent and oppressive" exploitation of animals in circuses.
It was named after Kimba, a lion which escaped from the Rony Roller Circus in the seaside town of Ladispoli, near Rome, in November.
Extraordinary videos showed the fully-grown lion with a shaggy mane walking through dark and deserted residential streets, before being captured and taken back.
His handler told AFP at the time he had posed no threat to the public.
More than 20 European countries have banned or heavily restricted the use of animals in circuses -- but Italy is not yet among them.
A law has been drafted but has been delayed, according to the LAV campaign group, which estimates that just under 2,000 animals are held in circuses across Italy.
Spraying washable paint or other non-toxic substances over famous memorials or paintings is becoming an increasingly common form of protest.
Activists on Sunday hurled soup at the bullet-proof glass protecting Leonardo da Vinci's "Mona Lisa" in the Louvre museum in Paris, demanding the right to "healthy and sustainable food".
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