Bayern Munich will be without Bastian Schweinsteiger and Franck Ribery in their DFB Pokal quarterfinal at Bayer Leverkusen on Wednesday night.
Ribery, who suffered an ankle injury nearly a month ago, also remains a doubt for the upcoming challenges in Champions League.
Schweinsteiger, 31, has been struggling with injuries ever since the 2014 World Cup final in Brazil. He only returned to Bayern's first team in December, and has since started in 14 games, coming off the bench in six other matches.
At the weekend in Dortmund, the Germany captain needed to be withdrawn in the second half with ankle problems following a challenge by Dortmund's Jakub Blaszczykowski early on in Bayern's 1-0 win.
"He won't play, definitely not," Guardiola told his news conference ahead of their cup tie at Bayer Leverkusen.
Schweinsteiger's absence only adds to Bayern's injury problems during the run-in of the season.
Arjen Robben, David Alaba and Javi Martinez are all unavailable for selection, along with Ribery, who is still struggling with an ankle injury sustained in the 7-0 Champions League win over Shakhtar Donetsk in mid-March.
Last week, the French winger admitted that he started training too early and was thrown off the course. However, the 32-year-old and the club were confident he could return to the squad against Leverkusen. This, however, is not the case.
"I don't know when he'll return. Ask the doctors," Guardiola said when asked about Ribery. According to a report in Bild on Wednesday, the Frenchman is also a doubt for the upcoming Champions League quarterfinal against Porto.
Still, Guardiola remained upbeat about Bayern's chances at in-form Leverkusen. "But I'm happy. There can be no excuses! It doesn't matter. It motivates me to find solutions," the Catalan coach said. "We have to change the way we play, at least a little."
Bayer Leverkusen have won their last five matches in the Bundesliga, scoring 13 goals and conceding none, ensuring the club are on course to qualify for Champions League once again, with an eight-point cushion over fifth-placed Schalke.
"I'm a big admirer of this team and their coach, and the match presents a huge challenge," Guardiola said.
"At this stage of the season it's about cool heads, big hearts and passion," he added, and praised his players for always being "up for it" despite the numerous silverware collected in the past couple of years.