Low bitumen recovery and poor froth quality are always encountered when processing poor oil sands using water-based extraction processes. Application of microbial treatment of the ore prior to bitumen extraction was proposed to resolve the challenges and develop a more versatile and effective extraction process. Microbial treatment was carried out by placing a poor processing ore in the culture solution with strain of Bacillus subtilis for a restricted period of time. Flotation tests showed that an improved bitumen recovery of 97% was obtained after the poor ore was microbially treated. The wettability of solids was found to be changed from hydrophobic to hydrophilic, leading to a great decrease of the long-range attractive force and adhesion between bitumen and solids. Saturate, aromatic, resin, and asphaltene (SARA) fraction analysis indicated that the content of heavy components of asphaltenes and resins was decreased with an increase in saturate and aromatic fractions after the microbial treatment of the ore. This was well-correlated with the rheological characterization of bitumen. The improved processability was attributed to the biosurfactant production in the culture solutions, alteration of solids wettability, and decrease in bitumen viscosity, which collectively promoted the liberation of bitumen from the solids surface, and gives a better oil quality.