Antoine Griezmann's second-half strike settled the Madrid derby at the Santiago Bernabeu on Saturday as a 1-0 defeat to Atletico all but ended Real's slim chances of claiming the title.
Griezmann produced an exquisite finish eight minutes into the second half as second-placed Atletico pulled further clear of Real in third.
Zinedine Zidane's men now trail leaders Barcelona by nine points ahead of the champions' visit of Sevilla on Sunday and the Frenchman acknowledged afterwards his side's title hopes were effectively over.
Diego Simeone's Atletico moved within five points of Barca, having become the first side in La Liga history to win three times in a row at the Bernabeu.
At the bottom, Sporting Gijon slipped into the relegation zone a day after fellow strugglers Las Palmas beat high-flying Eibar 1-0.
Abelardo's men went down 4-2 to Espanyol, and had Nacho Cases dismissed seven minutes from time.
Carlos Castro opened the scoring 20 minutes in at the Estadio Municipal El Molinon only for Buirgui to equalise prior to Gerard Moreno's brace before the hour.
Castro's second threatened a comeback but a Luis Hernandez own-goal following Cases' dismissal extended the hosts' winless run to five matches.
Getafe's slide down the table continued as they fell to a 1-0 loss at Celta Vigo - Nolito's first goal since returning from a thigh injury proving the difference - while Rayo Vallecano fought back from a slow start to draw 2-2 at Real Betis.
Ruben Castro scored twice for the hosts inside 29 minutes but a second-half brace from Manucho gained Rayo a point in Seville, with Real Sociedad and Malaga drawing 1-1 in the day's final game.
Imanol Agirretxe cancelled out Duje Cop's opener nine minutes from time as Sociedad's four match winning run in La Liga came to an end.