The maverick party, which took control of Rome in a landslide election victory in June, had always expressed doubts about staging the sporting spectacular, saying the heavily indebted city could ill afford the investment needed.
On Wednesday, party leaders told supporters at a rally near Rome that holding the games would encourage corruption and only benefit wealthy construction firms.
"People who want to get rich by spreading cement over our country can go to hell. It is for this reason that we will not accept the logic of the Olympics," said one of M5S's leading lights, Luigi Di Maio.
The other 2024 bidders are Paris, Los Angeles and Budapest. The International Olympic Committee is due to select the host city in September 2017.
Rome mayor Virginia Raggi, who is embroiled in controversy over the chaotic start to her rule, has made clear she has other priorities but has yet to definitively back away from the bid.
Italian Olympic Committee president Giovanni Malago has said the Rome bid would automatically fail without the backing of the city council. He is due to see Raggi in the coming weeks to try to convince her to support the project, which has been warmly endorsed by Prime Minister Matteo Renzi.
The man heading Rome's bid is veteran businessman and former Ferrari chief Luca Cordero di Montezemolo, who was manager of the Organising Committee for 1990 World Cup in Italy.
"We have to do these Olympics with Montezemolo?" asked another M5S leader, Alessandro Di Battista. The crowd in the town of Nettuno yelled 'no' in response.
"This is the same Montezemolo who organised the 1990 World Cup. We paid off the last debts for that in January," he said.
(Reporting by Crispian Balmer)
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