Penn won an Academy Award in 2008 for his role as gay activist and politician Harvey Milk in the biopic, which topped the poll of the most important movies depicting gay people in the last 10 years.
Officials at charity Stonewall, who conducted the survey, commended director Gus Van Sant's drama for its realistic portrayal of the homosexual community, reported Daily Star.
Milk's gay activist friend Cleve Jones, portrayed in the movie by Emile Hirsch, said, "This is a fantastic achievement for Milk.
"As someone who was depicted in the film, I feel very proud that gay people are saying it's the most significant film for them in the last decade - it shows that the campaign for full gay equality has reached a whole new generation of people, which gives me real hope."
Family drama 'The Kids Are All Right', starring Julianne Moore and Annette Bening as lesbian mothers, was in second place, while cowboy movie 'Brokeback Mountain' came third.
'A Single Man', starring Colin Firth as a gay university professor, and 'The Hours' rounded out the top five.
