Atletico Madrid, who are second, travel to second from bottom Sporting Gijon on Saturday while Real Madrid hope to keep their slim title hopes alive on Sunday by defeating fifth-placed Sevilla at the Bernabeu.
Barca, eight points clear at the top with nine matches left, could find their 38-game unbeaten sequence in all competitions seriously challenged by high-flying Villarreal.
"Our competitiveness and hunger gives us confidence for the road ahead," said defender Javier Mascherano. "To go where we want we have to be motivated for every game."
Barca beat Arsenal 3-1 at the Nou Camp on Wednesday to reach the Champions League quarter-finals 5-1 on aggregate.
Atletico needed penalties to see off PSV Eindhoven in their midweek Champions League tie and coach Diego Simeone may opt to rotate his squad for the trip to Gijon.
Simeone is without Diego Godin who has a muscle tear in his right thigh.
The injury will keep the centre back out for at least three weeks and threatens to weaken an Atletico defence that is the strongest in La Liga, letting in 12 goals all season.
Stefan Savic is doubtful for the Madrid club while Tiago remains a long-term injury absentee and Yannick Ferreira-Carrasco is suspended.
Karim Benzema could feature against Sevilla, with Real confirming the France striker came back to training on Wednesday after being out since Feb. 27 with a hamstring injury.
The 28-year-old has scored 19 goals in 20 La Liga games this season and his return is a boost for Real who are without suspended defenders Sergio Ramos and Pepe.
Real are four points behind Atletico and 12 adrift of Barcelona.
(Writing by Ed Dove; Editing by Tony Jimenez)
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