The dozen alleged mobsters are accused of the 1989 murder of Francesco Pepi and killing two other men, Rai News reported.
Pepi was reportedly killed for refusing to pay the Sicilian mafia protection money, known locally as the “pizzo”.
The breakthrough in the case came with the help of mafia informants, as part of a wider investigation into mafia wars in the southern province of Caltanissetta, Rai News said.
The businessman’s murder came during a violent period in Sicily's mafia history, when the government launched a crackdown on the criminal underworld, a move which the mafia fiercely fought against.
High-profile killings include those of anti-mafia prosecutors Giovanni Falcone and Paolo Borsellino, who died in separate bomb attacks in 1992.
The previous year the Sicilian mafia gunned down Libero Grassi, a business owner who, like Pepi, became known for refusing to pay protection money.
In recent years a number of organizations have been set up to strengthen the fight against the racket, with hundreds of businesses displaying “Addiopizzo” (Goodbye, protection money”) signs.
But the mafia still retains a tight grip on society in Sicily and elsewhere, using the racket to wield influence rather than as a main source of income. The main mafia group in Sicily, the Cosa Nostra (“Our thing”), has built an international criminal network profiting from drug trafficking and money laundering.