Jordan Goddard, brought on as a substitute in the 93rd minute of the game, was reported to police by a Corby Town supporter for relieving himself as he warmed up at the side of the pitch.
The incident was said to have taken place on a corner flag at the Corby end of Bristol's Memorial Stadium.
It allegedly happened just ahead of half-time in front of 500 fans during an FA Cup tie that Bristol won 3-1.
James Crawford, 48, was watching the match with his 12-year-old son.
He told the Sun: 'I've never seen anything like it. He was doing stretches, then put his hands in his shorts and pulled himself out.'
Fans jeered Goddard for his actions but rather than being embarrassed, the 18-year-old midfielder looked back and laughed.
An Avon and Somerset Police spokesman said: 'We are aware of it and we are looking into it in liaison with the football club.'
A spokesman from Bristol Rovers added: 'This is subject to a club investigation and will be dealt with internally.'
Former Argentinian goalkeeper Sergio Goycochea was well-known for his pre-penalty shoot-out ritual. In a bid to try and calm himself he would urinate on the pitch.
He explained: 'Until a match finishes you cannot leave the field of play. And if you have any natural human urges, you have to go on the field. 'So that is what happened in the 1990 World Cup quarter final against Yugoslavia.
I went and we won.'There was another shoot out in the semi final against Italy so I went again and we won again. It was my lucky charm and I went before every shoot out. I was very subtle, nobody complained.'
In April this year, Cheltenham Association Football League promised a clampdown on footballers urinating at the side of the pitch.However, manager Martin Gulliver said there was a simple explanation and that the players were 'responsible blokes' who, rather than peeing in the bushes were searching for a lost ball.
Managers from the league, however, issued a reminder to clubs that footballers shouldn't urinate in public.In October, referees officiating matches in the league were urged to red card players caught in the act.