A Spanish association representing open-source software users has filed a complaint against Microsoft to the EU.
The 8,000-member-strong Hispalinux, which represents users and developers of the Linux operating system in Spain, said Microsoft had made it difficult for users of computers sold with its Windows 8 platform to switch to Linux and other operating systems.
Lawyer and Hispalinux head Jose Maria Lancho said he delivered the complaint to the Madrid office of the European Commission this week.
Microsoft declined to comment and officials at the European Commission were not available.
In its 14-page complaint, Hispalinux said Windows 8 contained an "obstruction mechanism" called UEFI Secure Boot that controls the start-up of the computer and means users must seek keys from Microsoft to install another operating system.
The group said it was "a de facto technological jail for computer booting systems making Microsoft's Windows platform less neutral than ever".
"This is absolutely anti-competitive," Lancho said. "It's really bad for the user and for the European software industry."
The European Commission has fined Microsoft, the global leader in PC operating systems, €2.2bn (£1.87bn) over the past decade, making it the world's biggest offender of European Union business rules.
The Commission found in 2004 that Microsoft had abused its market leader position by tying Windows Media Player to the Windows software package and relations have remained tense.
The company took a more conciliatory approach in recent years, settling another antitrust investigation in 2009 related to the choice of a browser in its Windows operating system.
It also lodged its own complaints to the Commission about the business activities of rival Google.
But on 6 March, the Commission fined Microsoft €561m (£476m) for failing to offer users a choice of Web browser.