The churches will reopen when a broader Italian government crackdown on public gatherings expires on April 3, Cardinal Angelo De Donatis, the papal vicar for Rome, said in a statement.
Catholic faithful have been exempted from the obligation to attend Sunday Mass.
The Vatican had spent days resisting having to take the drastic measure of shuttering places of worship in the overwhelmingly Catholic country.
It closed its museums and even the Saint Peter’s Basilica – parts of its soaring dome designed by Michelangelo – to tourists as the death toll continued to mount.
All masses, weddings and funerals across the country have also been called off.
But some church buildings in the country will stay open as long as the faithful follow government regulations and remain a metre (three feet) apart while inside.
It was not immediately clear when Rome’s churches were last forced to close en masse.
The Nazis and Italian Fascists kept Pope Pius XII confined to the Vatican during World War II.
Some Rome churches kept their doors open during the war.
The closures come with the pope himself suffering from a cold and communicating with the faithful by livestream as a safety precaution.
Pope Francis complained of feeling “caged” while reading his traditional Sunday Angelus Prayer into a camera from a Vatican library instead of his usual window overlooking crowds on Saint Peter’s Square.
The 83-year-old was also forced to miss his weekly Wednesday appearance on the square that he often uses to hug and shake hands with the faithful from across the world.
The new regulations cover the Italian capital and not the Vatican City statelet located entirely within Rome.
The Holy See has recorded one COVID-19 infection and is awaiting the results of another person who attended one of its functions at the start of the month.
The cardinal’s statement said access to “churches of the Diocese of Rome open to the public – and more generally to religious buildings of any kind open to the public – is forbidden to all the faithful”.
The statement added that monasteries would remain open to “communities that habitually use them as residents”.
“This provision is for the common good,” De Donatis wrote.
The Italian government on Wednesday (Mar 11) announced a comprehensive crackdown that closed all stores except for pharmacies and groceries.
De Donatis said he was finally moved to close Rome’s churches by “the even more binding restrictions placed on the ordinary movement of people”.