An ally of the United States in its war against Iraq, Prime Minister Silvio Berlusconi and his ruling Forza Italia party boycotted the 24 km long march, claiming the event had been hijacked by left-wingers.
But nothing dampened the spirits of the marchers, who strummed guitars, banged drums and sang peace songs as they walked down the streets.
One section of the crowd was blanketed by a huge rainbow-coloured peace flag as they marched.
In a message to the marchers, Pope John Paul said Europe should become synonymous with peace.
Awared this year's Nobel Peace prize, Iranian lawyer Shirin Ebadi also delivered a message to the marchers via the independent Italian television channel La 7, which carried the event live.
"Yours is a road towards peace and the rights of man and of peoples," Ebadi said.
Marchers signed a petition demanding that Article 11 of the Italian constitution, which repudiates war as a way of resolving international conflicts,should be absorbed into the European Union's first constitution.
Vittorio Agnoletto, one of the leaders of the Italian anti-globalisation movement, said he was marching to protest against the involvement of EU states in Iraq.
"The administration in that country must first be put in the hands of the UN so that elections can be held under which these people can govern themselves," he said.