Ronaldo failed to convert his spot kick and Madrid fell short in its fight back to draw 2-2 with Valencia at home, letting Barcelona move four points clear after beating Real Sociedad 2-0.
Manchester United is set to end its one-year hiatus from the Champions League, while Paris Saint-Germain was also left in position to clinch the French league crown after Lyon stumbled.
Here is a look at the action in Europe's top leagues:
Neymar scored his 35th goal of the season and substitute Pedro Rodriguez netted from an acrobatic bicycle kick with five minutes left to secure Barcelona's win over David Moyes' Sociedad.
Madrid appeared set to keep pace after Gareth Bale and Ronaldo hit the woodwork early, but Paco Alcacer and Javi Fuego struck for Valencia before goalie Diego Alves smothered Ronaldo's spot kick in first-half injury time.
Pepe and Francisco "Isco" Alarcon leveled for Madrid in a frenetic second half at the Santiago Bernabeu that concluded with Valencia clinging to a draw that may cost the hosts the title.
Barcelona can dethrone Atletico Madrid with a win at its Vicente Calderon in the next round. If, and when, that match is played. The final two rounds are in doubt after the Spanish federation announced it was suspending all matches from next Saturday in protest at a proposed law regulating the sale of broadcasting rights.
After a second slip to fourth-place Valencia this season, Madrid's best chance for silverware depends on overturning its 2-1 loss against Juventus in their second-leg Champions League semifinal on Wednesday.
"It's very difficult for us now, but we have to wear these colors with pride, and try to win our games," Pepe said. "We have to pick ourselves up for Wednesday's match."
There was also drama in the fight to avoid relegation as Albert Lopo's last-gasp header to salvage Deportivo La Coruna a 1-1 draw at Athletic Bilbao altered three teams' place at the bottom of the table.
Deportivo, Eibar and Granada, which earlier beat already-relegated Cordoba 2-0, were left tied on 31 points. But the tiebreakers meant Deportivo escaped the drop zone, while Granada was left in 18th and Eibar fell to 19th.
Manchester United ended a three-match losing run to move within sight of Champions League qualification, while Burnley became the first team to be relegated from the English Premier League this season.
Marouane Fellaini scored a 78th-minute winner as United beat Crystal Palace 2-1 to move provisionally seven points clear of fifth-place Liverpool. Anything other than a win for Liverpool at champion Chelsea on Sunday and United is back in the Champions League.
The rest of the focus was on the fight to avoid relegation, which proved beyond Burnley despite a 1-0 win at Hull.
Wins for Sunderland, Leicester, and Aston Villa pushed them clear of the bottom three, and left Hull and Queens Park Rangers as the teams most likely to go down with Burnley.
Last-place QPR is nine points from safety with three games left, the first of which is away at Manchester City on Sunday when relegation could be sealed. Hull is third from bottom and two points from its nearest rival, Newcastle.
Borussia Moenchengladbach defeated Bayer Leverkusen 3-0 to go provisionally second in the Bundesliga while Bayern Munich lost 1-0 to Augsburg for the champion's fourth straight loss across all competitions.
Bayern hosts Barcelona for the second leg on Tuesday, hoping to turn around the 3-0 deficit.
"The league doesn't play a big role for us anymore. On Tuesday, we'll see if we can make it to (the Champions League final in) Berlin. If not, we'll celebrate the title," coach Pep Guardiola said.
Moenchengladbach ended fourth-place Leverkusen's 10-game unbeaten run, and moved one point above Wolfsburg, which visits relegation-threatened Paderborn on Sunday.
Roma lost 2-1 at AC Milan to leave it at risk of missing out on second place in Serie A, while Paul Pogba marked his comeback with a goal as newly crowned champion Juventus drew 1-1 against relegation-threatened Cagliari.
Massimiliano Allegri rested almost all his stars ahead of Wednesday's Champions League semifinal at Madrid, and it was Pogba who opened the scoring on the stroke of halftime, 52 days after a right-thigh muscle tear.
Luca Rossettini leveled five minutes from time to give Cagliari a crucial point. The Sardinian side moved to within five points of safety, with 17th-place Atalanta visiting Palermo on Sunday.
Lyon's hopes of winning the French league appeared over after slumping to a 3-0 loss at Caen, leaving the seven-time champions six points behind leader Paris Saint-Germain with only two games left.
Lyon's first defeat in six matches paved the way for PSG's third consecutive title.