The ecological and physiological factors governing microbial activity in the Southeast Vassar Vertz Sand Unit (SEWSU), Payne County, OK, an oil reservoir selected for a microbially enhanced oil recovery field pilot, were studied. Analysis of the brines from the reservoir showed that the SEWSU reservoir is a hypersaline environment rich in calcium and magnesium cations, and contains most of the inorganic nutrients required for microbial growth. Substantial amounts of sulfate and sulfide were detected in the brines, indicating a potential for sulfate reduction activity. Of the various carbohydrate‐based nutrients tested, a molasses‐ammonium nitrate nutrient mixture best stimulated the metabolism and growth of the microbial communities in the brines. Sulfide was not detected in any brine samples that received high levels of nitrate, even when additional carbon sources such as molasses were added. The addition of nitrate also resulted in shorter lag times, higher maximum turbidities, and larger pressure increases due to gas production. Aerobic heterotrophic bacteria, anaerobic heterotrophic bacteria, anaerobic molasses nitrate‐using bacteria, sulfate‐reducing bacteria, and methanogenic bacteria capable of growing at high salt concentrations were detected in all the brines. Several bacterial strains were isolated, five of which were further characterized. All five isolates were strictly anaerobic and obligately halophilic, requiring a minimum of 6–9% NaCl (w/v) for growth in complex medium. Glucose was fermented to HvCO2, ethanol, and acetate by all the five strains. In spite of the high salinity, the SEWSU reservoir contains an active microbial community whose metabolism and growth can be stimulated by the addition of carbohydrate‐based nutrients.