The party made the stunning gains in June in a stinging blow for Prime Minister Matteo Renzi and although M5S mayor for Rome, Virginia Raggi, has not publicly voiced her opposition, a blog by the party's founder Beppe Grillo on Sunday clearly said it was against the city bidding for the Games.
An editorial titled "The Games in Rome? No thank you!" said they would only serve to "condemn the future of youths to debt and act as a powerful arm of massive distraction".
The piece was written by Elio Lannutti, a party lawmaker and the head of a consumers' association. Raggi, Rome's first female mayor, has repeatedly pledged her absolute loyalty to the party's founder.
Raggi had continually repeated in her election campaign that the Games were not a priority for the Eternal City.
Rome is one of four candidates to host the Olympics in 2024 along with Paris, Budapest and Los Angeles. The quartet must submit the second part of their candidacy by October 7. Prime Minister Renzi on Saturday deplored the party's stand, and dismissed their contention that the money use to host the Games could be better spent.
"I am proud to be Italian but I am sad when my country refuses to take up a challenge that would please many, simply because of a few people," he said. "Either the money for the Games come to Rome or it goes to Paris," he added.