It ordered Apple to pay 134.5 million euros and e-commerce firm Amazon 68.7 million euros for infringing EU laws through restrictions which penalised sellers of Apple and Beats products.
US big tech firms have faced a slew of antitrust challenges in Europe -- an approach being watched closely by regulators in Washington, which has pledged to intensifying scrutiny of the technology industry.
The Italian watchdog said a 2018 deal between the two US companies had "barred official and unofficial resellers of Apple and Beats products from using Amazon.it, allowing the sale of those products in that marketplace only to Amazon and to selected parties in a discriminatory manner".
The aim had been to restrict the number of retailers and limit cross-border sales, it said.
The agreement spelled bad news for consumers, because at least 70 percent of electronics goods bought in Italy were purchased on Amazon, it added.
The watchdog said its investigation had attracted the attention of "the national competition authorities in Germany and Spain, which have also launched similar procedures".
A crackdown on Big Tech firms could lead to the breakup of the largest platforms, with Europe powering ahead with antitrust litigation and US lawmakers eyeing moves to make antitrust enforcement easier.
Big Tech critics in the European Union and United States want Apple and Google to loosen the grip of their online app marketplaces; more competition in a digital advertising market dominated by Google and Facebook; and better access to Amazon's e-commerce platform by third-party sellers.