The European Commission sent letters to makers of computer hardware and electronics but said they were confidential and would not name the companies.
In a statement, it said it wanted to "assess the conditions offered by Microsoft" for the software maker's licensing policies.
The "fact-finding process is at a very early stage (and) no further details will be supplied," the Commission added.
EU spokesman Tilman Lueders said the Commission's concerns focus on "licensing conditions under the competition rules."
Reacting to the EU move, Microsoft said its software licensing polices are already tightly supervised and should not trouble EU antitrust investigators.
The company's "agreements with hardware makers are subject to strict regulations and scrutiny as a result of legal proceedings in the United States," said a Microsoft official, speaking on condition of anonymity.
The official rebuffed suggestions of unfair business practices, saying its policies are "appropriate, pro-competitive and in compliance with the law."
The EU head office has been investigating the software giant's business practices for the past four years. Microsoft filed a response this month to rebut charges it was illegally trying to extend its dominance with Windows operating systems into markets for servers and multimedia players.
The four-year-old case is expected to result in fines, which could reach about $3 billion, as well as possible orders to disclose more of its prized software code to rivals and change how it sells Windows software. There is no deadline for a decision.