E Bar
41 Quebec Street, Guelph, Ontario
100% positive
Directions: Not physically accessible (so many stairs), however for many events they do make all of the washrooms unisex/gender neutral. Staff is supportive - if you have any issues I'm sure they would address them conscientiously. For events that have segregated washrooms by gender, there are other washrooms (closer to the cinema - if you walk through the greenroom towards the entrance above the bookshelf, there are washrooms right beside the cinema on the second floor). They tend to be a little more discreet and less busy than the main washrooms near the back of the venue.
Comments: Events like Fierce and many of the Femme Rebelles Burlesque shows have gender neutral washrooms. Check event listings or call ahead to make sure.