The Valencia forward pulled up with an injury just 33 minutes into his side's 3-0 win over Russia in the Euro 2008 semi-final.
Villa, who left the pitch in tears, was in pain after taking a free-kick, much to coach Luis Aragonés' displeasure.
The manager said after the game, "Villa will not be able to play in the final because he has pulled a muscle."
'El Guaje' is currently the tournament's top scorer with four strikes - three of which came against Russia in the group stages - but his crown is now under threat from Germany.
Forward Lukas Podolski has struck three times already, while Michael Ballack, Bastian Schweinstieger and Miroslav Klose each need two more goals to equal Villa's tally.
How Aragonés' side will line-up against Die Nationalelf is now unclear, but a showcase performance from Francesc 'Cesc' Fàbregas, who replaced Villa as a first half substitute, could see him retain a place in the first XI.
Steve Michaels, Goal.com