The Catalan outfit entered the match looking to make it eight straight wins at the start of their 2024-25 campaign, but they found themselves 2-0 down in the first half of the fixture and were unable to fight back despite scoring twice in the second period.
Ante Budimir registered twice for Osasuna, while Bryan Zaragoza and Abel Bretones were also on the scoresheet for the hosts, with Pau Victor and Lamine Yamal netting for the visitors.
Flick made a number of changes to his starting side, with the likes of Raphinha, Yamal, Alejandro Balde and Marc Casado left on the bench, and the German has taken the responsibility for the defeat.
However, the head coach admitted that he had no option but to make changes due to the number of players currently unavailable for selection due to injury problems.
Barcelona suffered first league defeat of the season to Osasuna
"We have to accept this loss. We didn't play well. I take responsibility," Flick told reporters after the contest with Osasuna.
"It wasn't a good game. We made a lot of mistakes, we made a lot of rotations to the team and maybe that's one reason why, but they were necessary, there are a lot of games and a lot of minutes accumulated.
"I told the team that I am responsible. We have an important match on Tuesday and we're on the right track. After the international break, many players will return, we will have more to choose from. The many rotations are no excuse, but we had no other option.
"It's my responsibility to take care of the players, and some of them had played too many minutes. Their rest was necessary.
"I'm very excited for the Champions League match. We need to get our first win, but it won't be easy. Young Boys are very fast and we'll need to deal with them."
Which players are currently out for Barcelona through injury?
Frenkie de Jong is now close to making his return from a long-term ankle problem, with the midfielder having a slight chance of being available against Young Boys in the Champions League on Tuesday night.
However, it is more likely that the Netherlands international will make his first appearance of the season in the La Liga contest with Alaves next weekend, when Barcelona will be bidding to return to winning ways in Spain's top flight.
Gavi is also making solid progress in his recovery from a long-term knee injury, but Marc Bernal and Marc-Andre ter Stegen are both out for the rest of the season with serious knee problems of their own.
Fermin Lopez, Dani Olmo, Ronald Araujo and Andreas Christensen are also currently on the sidelines, but all four should be available for selection before the end of 2024.
Barcelona will be under pressure to pick up a positive result against Young Boys in the Champions League, having suffered a 2-1 defeat to Monaco in their competition opener earlier this month.