Richmond's Prestia and Melbourne's May will front court next month after being charged over a fight in the Victorian coastal town of Sorrento last December.
Prestia is protesting his innocence after being charged with recklessly causing serious injury.
And May, who has been charged with affray, also denies any wrongdoing.
Prestia, a triple premiership player at Richmond who was a former teammate of May at the Gold Coast, confirmed he had been charged as "a result of an altercation that apparently took place on Boxing Day 2024 at Sorrento".
"I was not involved in the fight. I was not even physically present when it apparently took place," he said on Thursday in a statement issued by Richmond.
"I deny that I am guilty of these offences and will strenuously defend these charges."
Richmond said the club would offer support to Prestia ahead of his court appearance.
"Dion strenuously denies any wrongdoing and will defend himself accordingly," a club statement said.
"As with any member of the community, he is entitled to the presumption of innocence."
May, a Melbourne premiership defender and an All Australian, has told the Demons he was innocent of the charge.
"Steven has advised the club that he was not involved in the alleged incident and will strongly defend the charge in court," Melbourne said in a statement.
"The club will continue to support Steven throughout this process."
Victoria Police said the AFL duo were charged by Mornington Crime Investigation detectives.
Police alleged two groups of men were involved in an altercation on Point Nepean Road at Sorrento about 2am on December 27 last year.
"Two men were taken to hospital with non life-threatening injuries at the time," police said in a statement on Thursday.
"Detectives interviewed three men in May as part of their investigation."
May and Prestia were charged along with a 26-year-old man from Blairgowrie, who will face a count of affray.
May and Prestia, who started their AFL careers together at the Gold Coast Suns, will front Dromana Magistrates Court on November 27.
Prestia is a three-time premiership player with Richmond who won the club's best-and-fairest award in 2019.
He started his career with May at the Suns in 2011, moved to the Tigers in 2017, and has played 237 AFL games.
May, after making his debut with Gold Coast in 2011, captained the club from 2017-18 before joining the Demons for the 2019 season.
The premiership player and dual All Australian has played 251 AFL games, but faced an uncertain future at the club when told to explore trade options last month.
No clubs sought to trade in the key defender, who remains contracted at Melbourne until the end of next year.
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