The 'Ocean's Eleven' actor and Real Madrid striker were named on a list of 78 possible witnesses, including Clooney's Italian girlfriend Elisabetta Canalis as well as several other prominent TV personalities.
Others include Venezuelan beauty Aida Yespica, Argentinian showgirl Belen Rodriguez and senior government officials, including Italian Foreign Minister Franco Frattini.
"George Clooney is one of several people we have included on our list of witnesses as part of our case," the Daily Mail quoted a source on Berlusconi's defence team as saying.
"The list includes several personalities as well as cabinet ministers and they will all testify and prove that the prime minister is innocent of the ludicrous allegations made against him," the source added.
Clooney, who has a home on the shores of Lake Como near Milan, has been a guest at a fund raising dinner hosted by Berlusconi, but there is no suggestion he was involved in anything untoward.
"It seems odd since I've only met Berlusconi once and that was in an attempt to get aid into Darfur," said the actor.
Moroccan belly dancer Karima El Mahroug, who has allegedly been paid for sex by the Prime Minister when she was just 17 years old, had last month claimed to have had sex with Ronaldo for cash and that she then poured champagne over his head after she felt she was mistreated by him.
The allegation was immediately denied by the footballer as 'totally false', saying that he had never met her.
A judge will now decide if Clooney, Ronaldo and others on the list are relevant and should be officially called to testify.
If they are confirmed, then they will receive official notification from the court and will be expected to attend a future hearing when called.