Virginia Raggi, from the anti-establishment Five Star Movement, is seen as the favourite against Roberto Giachetti of the centre-left Democratic Party (PD).
Her victory would be a blow to Italy’s Prime Minister Matteo Renzi.
His PD party may also lose in Italy’s financial capital, Milan, and faces tough battles in Turin and Bologna.
Ms Raggi, a 37-year-old lawyer, won 35 per cent of the vote in the first round two weeks ago, against 24 per cent for Mr Giachetti.
The next mayor of Rome will find a city mired in debts of more than £10bn, - twice its annual budget.
Polls were scheduled to close at 11pm last night, with vote counting beginning immediately. Romans are impatient for change and improvement, especially of their strike-plagued mass transit service and undependable rubbish collection agency.
If Raggi wins, she will be the first woman to serve as Rome mayor.
Like that of challenger Roberto Giachetti, a Democrat, Raggi’s campaign was short on details for a Rome renaissance.
Her opposition to the bid for the city to host the 2024 Olympics is a direct challenge to Renzi’s embrace of the possibility.
An Olympic gold-medalist, swimmer Federica Pellegrini said she hoped Italy would still pursue the 2024 games.
“I hope that Rome, and with Rome, I say all of Italy, and this must be clear, continues with the candidacy,” Pellegrini, who will be Italy’s flag-bearer in this summer’s games in Brazil, told the Rome daily Il Messaggero.
She added that while she wasn’t talking politics, “I believe that every city should be livable without putting into doubt such an important candidacy.”
Raggi’s rival Giachetti served as a mayoral aide in an administration pre-dating the years of City Hall patronage and kickback scandals.
Since neither candidate won more than 50 per cent of the vote, yesterday’s runoff between the top two vote-getters was necessary.
The 5-Star Movement, founded by comic Beppe Grillo, also has a candidate in Turin’s runoff.
The electoral duel in Milan, Italy’s financial heart, saw Renzi’s clout on the line.
As Democratic Party leader, Renzi campaigned enthusiastically for the centre-left candidate, Giuseppe Sala, who successfully carried off the recent international Expo in that city, a point of pride for the premier. The challenger, Stefano Parisi, backed by the center-right, has a similarly solid curriculum as a manager.
Other major cities holding runoffs included Naples and Bologna.
Voters in Rome voiced their disenchantment with past local politicians.
“The outskirts are abandoned. The historic city centre is just a showcase for tourists,” said Simonetta Facioni, after casting her vote.
Some of the dozens of local politicians and businessmen arrested in the scandal are on trial on charges including corruption, graft and using Mafia-like intimidation methods to win municipal contracts.
An investigation by prosecutors revealed that many contracts were awarded without bidding to cronies of City Hall officials, including from the Democrats as well as from right-wing and other parties.