Under IOC rules, host cities are not permitted to criticise each other's bids. Spain's Olympic vice-president Jose Maria Odriozola - who is not formally part of the Madrid bid organising team - labelled the Brazilian city as "the worst of the four candidates".
Odriozola was also quoted by Spanish news agency Efe as saying Rio has security issues, and its status as a favourite was based on “marketing and sentimentality”.
"His comments are totally unacceptable, and we are lodging a formal complaint to the Ethics Commission," said a Rio 2016 statement. "This gentleman is clearly out of step with the report of the IOC's own Evaluation Commission."
Madrid 2016 chief executive Mercedes Coghen apologised for the remarks at a ceremony to launch the Spaniards' arrival in Copenhagen.
Rio also complained to the IOC ethics committee earlier this week following remarks from Chicago mayor Richard Daley, who said that hosting the 2014 football World Cup was not the same as hosting the Games.
Rio, Madrid, Chicago and Tokyo are all lobbying in Copenhagen ahead of Friday's vote by the IOC.
