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Television cameras captured fans in the stands making gestures with their arms while also chanting.
Real winger Peter Gonzalez appears to have been subject to 'monkey chants' as well as defender Marvel after being booked.
Fans also chanted Retiro Park was 'Spanish, not Ecuador,' and Peter made sure to not react to the crowd.
Atletico fans doing the nazi salute during yesterday's youth league game. Shocking. pic.twitter.com/bfdOw1cH4b— James Walker (@William41874992) March 4, 2022
Atletico won the game 3-2 and will face German outfit Borussia Dortmund in the quarter-finals.
Pablo Barrios bagged a brace to send current holders Madrid out of the tournament.
The game was marred by the off-pitch incident and fans have expressed their disgust online.
One commented: "This is disgusting."
Another said: "They do not speak for our club."
While third put: "How can they do this?"
Former Atletico striker Fernando Torres is currently in charge after taking the job last summer and he's doing a fine job on the pitch.
Armando de la Morena, an ex-coach at Atletico, believes Torres is already a great manager and can achieve more in his career.
"A coach like Fernando, knowing his career a bit, as a player and always with the respect that he has acted, has the conditions to be a great coach," Morena explained to AS.
"Because of how he expresses himself, the serenity that he has, the calm.
"I know it from the players too, who tell me wonders about him, how he transmits, how he gives them the concepts."
Atletico's first-team are having a tricky season after winning La Liga last time out.
Diego Simeone's men sit fifth and are 15 points behind leaders Real Madrid.
They're in the last 16 of the Champions League and are currently tied 1-1 with Manchester United.
Atletico will travel to Old Trafford for their second leg tie on March 15.
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