The three-time former premier claimed the task of cleaning up the scandal-hit capital would be too much for far right candidate Giorgia Meloni, who is due to give birth soon after the election in May or June.
"A mother cannot dedicate herself to a terrible job and running Rome today is terrible job which will require 14 hours a day in the office," Berlusconi said.
The media tycoon, famed for his bunga bunga parties with young female escorts, was speaking in support of Guido Bertolaso, who said yesterday that Meloni would be unable to "sort out the rubbish, the traffic jams and the rats in Rome when she will have to be at home nursing."
Behind the row lies a battle for ascendancy on the right of Italian politics with Berlusconi backing Bertolaso and Northern League leader Matteo Salvini endorsing Meloni in an election race the right feel they can win.
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The last mayor, the centre-left's Ignazio Marino, was forced to resign last year after a minor expenses scandal intensified pressure on him over his perceived mismanagement of the cash-strapped capital.
Female politicians lined up to denounce the outbursts as symptomatic of a lingering macho culture in Italian politics.
"When will they ask a male candidate to withdraw because he is not telegenic? Or because he is due to be a father?," asked Constitutional Reform Minister Maria Elena Boschi on Twitter.