The Spaniard joked he would buy journalists "a big class of beer" if the 28-year-old England forward did not start in the return clash at the Allianz Arena, after Rooney returned to training on Tuesday following a toe injury.
"He is going to play - 100%. I bet you a big glass of beer he is going to play," Guardiola said.
"The big players have to play. Rooney is one of the best players I've ever seen in my career so if he doesn't play in this game, it means he has a lot of problems.
"I believe he will play and Antonio Valencia too, because I am certain Bastian Schweinsteiger (suspended) is not going to play."
Wednesday's game is delicately poised at 1-1 following the first leg at Old Trafford and United have a real belief they can pull off a shock result in the German capital and progress to the semi-finals.
But Guardiola, the former Barcelona manager, believes he has figured David Moyes out tactically.
"When they play at home they attack a little bit more, when they're away they defend and wait for the counter-attack," he said.
"Maybe I am wrong. Maybe they are thinking something different. But I think they are going to wait at the back, with eight or nine players in the box, and wait for our mistake or a chance at a free-kick or corner to punish us."
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