Favio Lotti, one of the demonstration's organisers, told Al Jazeera: "The world does not need another military base. It needs greater peace efforts by the US and by the Italian government."
Lotti said that claims by those in favour of the base that it would benefit the economy were false.
"The Italian government is contributing to one third of the expenses of the base," he said.
Protesters are also concerned that the expansion of the base could cause traffic, deplete local resources including water and gas, and increase the risk of Italy being targeted by a terrorist attack.
Romano Prodi, Italy's prime minister, angered allies last month when he decided not to renege on a pledge by Silvio Berlusconi, the former prime minister, to allow the expansion of the base in Vicenza.
"Demonstrations are the salt of democracy, but they should be peaceful, serene and without violence."
Italian prime minister
Prodi has said his government had no reason to halt the expansion, which has been approved by Vicenza's city council.
The Italian leader called on the protesters to remain peaceful.
He said: "Demonstrations are the salt of democracy, but they should be peaceful, serene and without violence."
The US embassy has put out a travel advisory urging US nationals to stay away from Vicenza on Friday and Saturday.
But Oscar Mancini, the Vicenza regional representative for Italy's largest labour union, the CGIL, said at a pre-march rally on Friday: "The mass media are calling us extremists. They haven't understood anything about Vicenza."
Achille Variati, Vicenza's former mayor said the protests were also misconstrued as anti-American.
He said: "It's about us against the city administration. It would be a big error to be anti-American."
The base is one of several issues that are troubling US-Italian relations.
After winning elections in May last year, Prodi withdrew Italy's troops from Iraq in December.
On Friday, a Milan judge ordered 26 US citizens, most of them believed to be CIA agents, to stand trial for the 2003 abduction of an Egyptian imam.
"I don't want any more Americans here and I don't want a new base. They should just leave us alone," said Pucci Mori, a resident of Vicenza who lives near the proposed base expansion.
"Wherever they go in the world, Americans cause trouble."