Bitumen extraction from the oil sands of Alberta has resulted in millions of cubic meters of waste stored on-site in tailings ponds. Unique microbial ecol- ogy is expected in these ponds, which may be key to their bioremediation potential. We considered that direct culturing of microbes from a tailings sam- ple as biofilms could lead to the recovery of microbial communities that pro- vide good representation of the ecology of the tailings. Culturing of mixed species biofilms in vitro using the Calgary Biofilm Device (CBD) under aerobic, microaerobic, and anaerobic growth conditions was successful both with and without the addition of various growth nutrients. Denaturant gradient gel elec- trophoresis and 16S rRNA gene pyrotag sequencing revealed that unique mixed biofilm communities were recovered under each incubation condition, with the dominant species belonging to Pseudomonas, Thauera, Hydrogenophaga, Rho- doferax, and Acidovorax. This work used an approach that allowed organisms to grow as a biofilm directly from a sample collected of their environment, and the biofilms cultivated in vitro were representative of the endogenous environ- mental community. For the first time, representative environmental mixed spe- cies biofilms have been isolated and grown under laboratory conditions from an oil sands tailings pond environment and a description of their composition is provided.