A video has emerged on social media showing a fan on the top level of Marvel Stadium spitting on fellow patrons below him.
The footage reveals two male spectators appearing to be joking, before one of them spits down onto the level one crowd.
The duo appear to immediately flee afterwards to head for the exit.
The AFL has reportedly been made aware of the incident and contacted for comment.
The video comes in the wake of fans behaviour at the footy being a major talking point last week.
League CEO Gillion McLachlan issued an open apology for the increase of security on match day making spectators feel uncomfortable.
“I want to be clear about this – our philosophy on this has not changed,” he said.
“No-one at the AFL is trying to stop our crowd being passionate, our fans barracking – no-one.
“It is devastating to think that our fans believe that something has changed, that is trying to limit their enjoyment at the footy.
“I won’t apologise for making footy a safe place, but we need to ensure we get the balance right.”