The competition watchdog fined the Milan-based broadcaster 51.4 million euros, while Sky Italia, whose largest shareholder is media tycoon Rupert Murdoch's 21st Century Fox, was fined 4 million euros.
The two broadcasters were accused of creating a cartel to carve up the rights to show matches of the top eight Italian teams in the 2015-2018 seasons, squeezing out competitors.
Mediaset, controlled by former Italian prime minister Berlusconi, said it was shocked at the decision and would appeal, asking for an urgent suspension. Berlusconi also owns the AC Milan soccer team.
Sky acknowledged the regulator's ruling. Ahead of reading the full text, it said the watchdog had recognised Sky was not the instigator of any anti-competitive agreement.
The auction of the TV rights for Serie A soccer, worth nearly 1 billion euros a season, had been delayed due to protests from rival bidders about the lack of transparency.
The watchdog launched its investigation in May last year, after the offices of the Milan-based broadcaster and satellite TV group were raided as part of a broader inquiry.
A first stage of the investigation was closed in December and the parties involved were called to respond to the findings before a panel in February.
Earlier this month, Mediaset sold pay TV unit Premium to French media giant Vivendi.
The antitrust authority also fined Lega Calcio, which manages Serie A, 1.9 million euros and its Swiss-based adviser Infront 9 million euros.
Infront, which said the ruling was based on a wrong and partial reading of events, said it would appeal. Lega Calcio denied any wrongdoing.
Mediaset shares were down 1 percent in early trade.(This version of the story was refiled to add dropped word in second paragraph)
(Additional reporting by Giancarlo Navach and Elvira Pollina; Editing by Mark Potter and Keith Weir)
