Prestia and Melbourne's All Australian defender 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.
"I have today been charged with the offense of recklessly causing serious injury as a result of an altercation that apparently took place on Boxing Day 2024 at Sorrento," Prestia said in a statement issued by Richmond.
"I was not involved in the fight. I was not even physically present when it apparently took place.
"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."
Melbourne's May, charged with affray, has yet to comment publicly.
Victoria Police said the AFL duo, former teammates at Gold Coast, 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 Gold Coast, 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.
17°C