Any traveller who has been in the US within the past 14 days must produce evidence of a negative test result taken within three days of arrival, regardless of vaccination status, said the Italian authorities.
Unvaccinated travellers from the US must also quarantine for five days and then be tested again before they are released.
On 21 June, Italy had changed the rules to allow US travellers to enter without quarantining, as long as they could present a negative test result, proof of vaccination or proof of recovery.
Italy is imposing the same restrictions on Japan, Canada and Israel, all of which have high case rates at present.
The new rules will be in place until at least 25 October.
The news comes a day after it was announced that Italy is dropping the five-day quarantine requirement for fully vaccinated British travellers.
On Monday, the Financial Times reported that the US rate of infection had risen to more than 300 new Covid cases per 100,000 people in the past 14 days, citing figures from the Centers for Disease Control and Prevention.
According to Johns Hopkins University, the US has led the world with the highest number of infections over the past 28 days, with a total of 38.8 million cases and 637,930 deaths.
The majority of EU countries opened their borders to American citizens in June; no other European countries have announced changes to their travel rules so far.