Millwall and the Football Association are investigating after match officials reported an alleged incident of homophobic abuse at The Den.
The Lions' 2-0 win over Reading on Saturday was briefly halted in the first half by referee Keith Stroud.
Stroud spoke with both managers after an incident at The Den was reported by one of his assistant referees.
An announcement was also made over the ground's public address system at half-time in line with advised protocol.
Stroud appeared to be called over by his assistant in front of the Dockers Stand as Reading full-back Tyler Blackett prepared to take a throw-in in front of a section of home supporters.
Millwall confirmed in a statement that "a comprehensive investigation is under way".
The statement continued: "The club has a zero-tolerance policy against discrimination of any kind.
"Any individual found guilty of such abuse at The Den is issued with an immediate lifetime ban."
After the game, Reading boss Mark Bowen said the referee had been clear in his handling of the incident.
"His words to me were, 'it's my job to make you aware that I've been informed that there were homophobic chants on the far side'," Bowen told BBC Radio Berkshire.
"He'd been made aware by the assistant on that touchline - that's all I know at this stage.
"It's a shame, it's probably happening too much now, but it's for Millwall to deal with."
Millwall manager Gary Rowett backed the club to handle the incident appropriately.
"As always, the club will do a thorough job," he told BBC Radio 5 Live. "I don't know what comment was made or indeed what was alleged.
"I don't necessarily think it was a chant, I think the linesman's heard a comment. There can be lots of comments in a game like that.
"The linesman, with what he's suggested the comment was, has to report it to the referee, who then in turn has to report it to the side of the pitch."
Millwall were fined £10,000 last year for racist chanting by some of their fans during an FA Cup fourth round win against Everton.
The club were told to "implement an action plan" after it was found to have "failed to use all due diligence" and ensure its supporters refrained from using racist language.
There have been a number of instances of homophobic chanting in English football recently, with the Metropolitan Police investigating following Chelsea's game with West Ham last month.
Everton also launched an inquiry into abuse aimed at Chelsea supporters and League One club Bristol Rovers were fined £7,500 and ordered to implement an action plan for failing to ensure spectators conducted themselves in an orderly fashion at a Carabao Cup tie against Brighton in August.