Real Madrid inflicted a second painful defeat on stuttering Barcelona in five days when Sergio Ramos nodded a late winner in a 2-1 success for Jose Mourinho's side in La Liga on Saturday.
Lionel Messi equalled Alfredo Di Stefano's scoring record for 'Clasico' matches of 18 goals with an 18th-minute leveller after Karim Benzema's opener for hosts Real in the sixth minute but it was scant comfort for leaders Barca, dumped out of the King's Cup by their arch-rivals 4-2 on aggregate on Tuesday.
Third-placed Real's victory does little to affect the title race, leaving them 13 points behind leaders Barca with 12 matches left, but is another big psychological boost ahead of Tuesday's Champions League last 16, second leg at Manchester United.
Real's trip to Old Trafford - last month's first leg at the Bernabeu finished 1-1 - is the club's overwhelming priority and Mourinho left key players including top scorer Cristiano Ronaldo and midfielders Xabi Alonso and Mesut Ozil out of his starting lineup for Barca's visit.
It was a strangely low-key affair for a clash between the Spanish giants and both teams appeared content to settle for a 1-1 draw until Ramos leaped to head home from a Luka Modric corner in the 82nd minute.
The Barca players were furious when their penalty appeals for an apparent Ramos foul on Adriano were waved away in the dying seconds and goalkeeper Victor Valdes was shown a yellow card and then a red moments later for furiously berating the referee after the whistle.
"Winning the league was more impossible when we were 16 points behind but as long as it's not mathematically ruled out Madrid has the obligation to keep fighting," Ramos said in an interview with Spanish television broadcaster Canal Plus.
"After two consecutive 'Clasico' victories we are going to Manchester convinced we can get through, but without being disrespectful to United."
Barca were looking for revenge after Real ended their treble hopes in Tuesday's Cup semi-final second leg but fell behind when Benzema was left unmarked at the far post and clipped an Alvaro Morata cross into the net.
Messi equalised when he got the better of Sergio Ramos and sent a low shot past Diego Lopez for his 50th goal of the season in all competitions.
It was the 16th straight game he has scored in La Liga, taking his tally to 39 for the season, and his 18th goal in his 25th appearance against Real, equalling Di Stefano's record set in the 1950s and 60s.
Ronaldo came off the bench with about half an hour left and the in-form Portuguese stung Valdes's hands with a powerful free kick before Morata was denied by the Barca keeper in a one on one after he was played through by Pepe.
After Ramos nodded the home side in front, Ronaldo curled another free kick against the post, narrowly failing to score an 11th goal in his last 11 'Clasicos'.
Although they are still 12 points clear of second-placed Atletico Madrid, who play at fourth-placed Malaga on Sunday, the defeat was another bitter blow for Barca, who had not lost at the Bernabeu in the league in the last four seasons.
Mourinho and his players now appear to have gained the upper hand over their great rivals, a significant turnaround from the Portuguese coach's early days in Spain when Barca were close to unbeatable under Pep Guardiola.
The Catalans have won only one of the last eight 'Clasicos' and have not tasted victory in five, their worst run since 2006-2008 when Frank Rijkaard was in charge.
Mourinho's record against Barca as Real coach is now five wins, six draws and six defeats in 17 official matches.
"We didn't play a bad match and I think a draw would have been a fair result," Barca defender Gerard Pique told Canal Plus.
"We felt it was a clear penalty but we can't use the referee's decisions as an excuse and we have to do enough to win games without relying on that.
"It's worrying because Barca is a club that always has to win but I have a lot of confidence in this team and I am sure we will get through it."
Barca have a La Liga game at home to bottom side Deportivo La Coruna on March 9 before they seek to overturn a 2-0 deficit when they host AC Milan for their Champions League last 16, second leg on March 12.