Prosecutors in Florence opened the probe after a mafia boss was caught on wiretap last year implying to a fellow inmate that the media magnate had urged him personally to commit acts of violence against the state.
It is the third time that the ex-premier has been investigated for a possible role in the mafia bombings in Rome, Milan and Florence that killed 10 people. The first two probes were dismissed due to lack of evidence.
The attacks were seen as revenge for the arrest of boss Toto Riina and the state's creation of harsh anti-mafia laws.
Giuseppe Graviano, serving life for his part in the bombings as well as the murder of two anti-mafia judges and an anti-mafia priest, claimed Berlusconi encouraged the violence for his own political ends, hoping to oust the old guard.
The ageing billionaire's right-hand man Marcello Dell'Utri served as the intermediary, according to a mafia hit man.
The co-founder of Berlusconi's Go Italy (FI) party was sentenced in 2014 to seven years in jail for mafia complicity.
Lawyer Niccolo Ghedini dismissed the latest probe as "the umpteenth investigation that will shortly be shelved, like the previous ones, as there is no new element and it has nothing to do with Berlusconi".