Eto'o double rescues Barca at Betis
Barca have 60 points from 23 matches while Valencia climbed to third on 38 after a 1-1 home draw with Malaga, who moved above Atletico Madrid into sixth spot.
Sevilla, in fourth, visit Espanyol on Sunday on a run of three straight defeats and Atletico host city rivals Getafe.
Barca have struggled at Betis in the past decade, winning twice in 10 matches and losing five including a 3-2 defeat last season in a game they led 2-0.
Guardiola left Lionel Messi and Thierry Henry out of his starting line-up and Betis took advantage, stunning the visitors with two goals in 25 minutes.
Defender Melli beat Sergio Busquets to a corner and sent a looping header past goalkeeper Victor Valdes. A defensive lapse then let Mark Gonzalez in to double the home side's lead.
Sergio Garcia's cross from the right was allowed through to the Chilean forward and he volleyed low across Valdes into the bottom corner.
Barca pulled one back in first-half injury time when Andres Iniesta went to ground in the area under a challenge from Juande and the referee pointed to the penalty spot, even though replays showed there was little contact.
Ricardo Pereira blocked the first effort by Eto'o but the Cameroon striker drove home the rebound.
Ricardo produced stunning saves to deny substitute Henry twice and Gerard Pique once as Barca pressed for an equaliser.
With the visitors pushing forward, Ricardo Oliveira had a chance to make it 3-1 when he went clean through but Valdes blocked the Brazilian's weak effort.
Eto'o grabbed his second double in consecutive weeks with a smart finish through the legs of Fernando Vega with six minutes left, the 23rd goal of the season for the league's top scorer.
"We were pushing for the win and I have no reason to reproach my players," Guardiola told reporters. "It was a good performance against a very good team."
David Villa put Valencia in front against Malaga with a clinical finish just after the break, the Spain striker picking up David Silva's pass and curling a shot over keeper Inaki Goitia.
The lead lasted six minutes as Malaga broke swiftly and Portuguese midfielder Eliseu drove in a low cross from the right that deflected into the net off defender Raul Albiol.
Osasuna extended their unbeaten run to four matches with a 0-0 draw at eighth-placed Deportivo Coruna but the Pamplona-based club remained 17th, one point above the relegation zone.