The center-right candidate, radio host and lawyer Enrico Michetti, is backed by an alliance of the far-right Fratelli d'Italia (FdI), Matteo Salvini's right-wing Lega party and center-right Forza Italia.
History professor and former finance minister Roberto Gualtieri is the candidate for the center-left Partito Democratico (PD).
The run-off -- taking place Sunday and Monday -- comes after there was no outright winner in polls two weeks ago.
Last week, the granddaughter of Italian fascist leader Benito Mussolini, Rachele Mussolini, won a second term as a city councilor in Rome in the October 3-4 municipal election.
Standing for Fratelli d'Italia, she won more than 8,200 votes -- the highest number of votes for any candidate -- and a huge increase on the 657 votes she received in the 2016 ballot.
Fratelli d'Italia, descends from the neo-fascist conservative MSI party -- or Italian Social Movement party -- and is led by Giorgia Meloni.
Polls close at 3 p.m. local time (9 a.m. ET) on Monday.
The run-off comes amid political tensions over the country's new "Green Pass" that mandates all workers are either double vaccinated or able to show proof of a negative Covid-19 test or recent recovery.
Protests erupted and turned violent in Rome on October 9 over the pass, which came into force on Friday. It is the strictest such mandate for workers in Europe.
On Saturday, a rally against fascist movements was held by Italy's national trade unions in the city's San Giovanni square.