Alvaro Morata scored twice to help Atletico Madrid to a 3-0 win over relegation-haunted Mallorca and stretch their unbeaten La Liga run to 12 games, as the victory consolidated their third place on 62 points to stay well on course.
The result gives Real Madrid the chance to move four points clear at the top of the table if they can overcome Getafe at home, a formidable lead with five games left.
Atletico advanced 4-2 on aggregate in the round of 16, thanks to two goals by Marcos Llorente in extra time and another by fellow substitute Alvaro Morata.
Messi's strike saw Barca return to the top of the standings with 31 points after 14 games, ahead of Real Madrid on goal difference, while Atletico were left in sixth spot on 25 points, having played one match more than the two pacesetters.
Unbeaten Real are a point ahead of promoted Granada and Atletico Madrid after Diego Simeone's side failed to break down their well-drilled local rivals.
Real Madrid climbed to the top of the standings with 14 points from six games, one point and one place ahead of Atletico Madrid, whom they face on Saturday in the derby at the Wanda Metropolitano.
The Argentinian playmaker's withdrawal will be a major concern for Barca, particularly given this was Messi's first start of the season after just recovering from a niggling calf injury sustained at the start of August.
Barca won a penalty early in the second half when Luis Suarez was fouled and Messi converted to put the league leaders in front and the Uruguayan forward made sure of the points with a simple finish from close range in the 82nd minute.
Atletico Madrid took a huge advantage in their heavyweight Champions League last-16 tie with Juventus, earning a 2-0 home win.
Messi scored Barcelona's second goal against SD Eibar shortly after halftime to reach a milestone in the competition after 435 league matches.
The UEFA Champions League round of 16 will see three England-Germany clashes and a true test of the Premier League v Bundesliga.
Real Madrid were playing their first competitive game since all-time top scorer Cristiano Ronaldo left and Julen Lopetegui succeeded Zinedine Zidane as coach.
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