The Atletico Madrid forward has suffered a hamstring strain and is unlikely to be fit for the first leg of the Copa del Rey semifinal against Los Blancos.
The attacker suffered the injury in Sunday's 4-0 La Liga win over Real Sociedad and was withdrawn from the game four minutes before the break, having opened the scoring at the Vicente Calderon.
The club confirmed Monday that the former Barcelona forward sustained damage to his left hamstring.
"David Villa suffered an elongation/strain without rupture (grade I) of the bicep femoris muscle on the back of the left thigh," the club stated on its Twitter account.
Atletico has not set a time frame on Villa's return to action, although he will likely miss Wednesday's Copa del Rey semifinal first leg clash with Madrid.
Villa has 13 goals in all competitions since moving from Barcelona in July.