The centre-right had most to cheer about, leading the field in Lecce, Verona, Padova and the northern port city of Genoa, which has been run by the left for the last 50 years.
5-Star had been hoping to build on its success a year ago, when it took charge of Rome after winning a landslide.
The political climate ahead of the vote became febrile this week after a deal on electoral reform among the main parties broke down amid bitter recriminations.
Moreover, in numerous contests, the main parties have taken a back seat and chosen to camouflage themselves in broad "civic list" coalitions rather than present their own individual candidates.
Center-right, center-left or independent mayoral hopefuls looked set to contest the run-off elections.
Candidates from the party founded by Beppe Grillo failed to make it to the run-off mayoral vote in any of the main contested cities at the weekend.
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She inherited a 17-seat majority in the Commons, but called the snap vote to take advantage of opinion polls putting her on course for a landslide.
The local races "put the brakes on the Movement's" rise, headlined La Stampa, the daily in Turin, a major city where only a year earlier the triumph of the populists' candidate there for mayor fueled 5-Stars' national ambitions.
5-Star is running neck-and-neck with the PD nationally, according to opinion polls, but it often struggles in local elections due to its loose organization and lack of high-profile candidates and is expected to score few successes on Sunday.
It also took control of a string of smaller municipalities.
Analysts said the party was punished, in part, for its poor track record in Rome, after winning elections there last summer.
But the talks collapsed in acrimony on Thursday, meaning the election is likely to take place as scheduled early next year.
Italy was due to hold elections in 2018, however, on December 6, 2016, Italy's then Interior Minister Angelino Alfano said that the country could hold snap parliamentary elections in February 2017, following the resignation of the country's Prime Minister Matteo Renzi in the wake of the government-proposed constitutional referendum.