Monkey chants were reportedly directed at Tottenham players Jermain Defoe, Aaron Lennon and Andros Townsend during the 0-0 draw at White Hart Lane on Thursday which was attended by UEFA President Michel Platini.
Lazio was charged after UEFA received the reports from referee Ovidiu Alin Hategan and match delegate Adonis Procopiou.
"UEFA has opened disciplinary proceedings against S.S. Lazio for the improper conduct of the club's supporters (racist behaviour)," European football's governing body said in a statement.
When the case is heard on Oct. 18 by UEFA's control and disciplinary body, Lazio could be facing a fine.
On Friday, Bulgarian club Levski Sofia said it has been fined €30,000 ($39,000) by UEFA for racist abuse by their fans at a Europa League match in July against Bosnian team Sarajevo.
But Piara Powar, executive director of the Football Against Racism in Europe group, wants clubs to be ordered to play their next game behind closed doors if their fans are found guilty of racism.
"There needs to be quite a hard symbolic action taken when these instances occur," Powar said.
A report by British legislators this week urged football authorities to do more to combat racism in football.
While the legislators praised the improvement in atmosphere at matches compared to the 1970s and '80s, players are now often the target of racist abuse online.
Chelsea on Thursday asked police to investigate abuse directed at midfielder John Obi Mikel on Twitter after the Nigeria international's mistake in Wednesday's Champions League opener against Juventus led to his team conceding an equalizer.
"The social media world, especially Twitter — because it's anonymous and so on — it's a dangerous vehicle to express yourself," Chelsea manager Roberto Di Matteo said. "I don't think it's fair. If somebody has to express his opinion, he should put his face to it."
The club wants the police to take "the strongest possible action" against Mikel's Twitter abusers.
"There's a lot of campaigns against (racism) and we have to try our best to fight it in every possible way," Di Matteo said. "We should try to make the people understand that it hurts other people, chanting and things like that."
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