Ecuador have lodged a complaint with World Cup organizers after local supporters gathered outside their team hotel and made noise ahead of their last-32 match against co-hosts Mexico.
Local media said roughly 1,000 Mexico fans staged a loud “serenade” in Mexico City on Monday.
The game is scheduled for Wednesday (02:00 BST) at Mexico City Stadium.
Supporters used noisemakers, pans, and buckets while singing the folk classic “Cielito Lindo” before police pushed the crowd back.
Ecuador’s football federation said the behavior ran counter to the World Cup’s ideals of fair play, equality, and unity, and added the team would do its talking on the pitch.
Mexico have won all three of their World Cup matches at Mexico City Stadium, while Ecuador’s upset of Germany secured their place in the knockouts.
The federation also expressed hope that such unsporting incidents would not spoil a football celebration between two friendly nations.
England, who face DR Congo on Wednesday (17:00 BST), would meet Mexico in Mexico City if both sides win their last-32 ties.
Fifa has been asked for comment.