Modification of oil-brine-minerals interfacial properties with biosurfactant-producing microorganisms and their extracellular metabolites has been considered as one of the viable strategies for microbial enhanced oil recovery (MEOR). In this study, the effect of lipopeptide biosurfactant produced by Bacillus subtilis on the interfacial tension (IFT) and wettability in oil-brine-mineral systems was quantitatively examined by monitoring dodecane-brine IFT and the contact angle of a dodecane-brine-quartz system during cultivation of B. subtilis. The effect of high temperature (35-45 degrees C) and pressure (similar to 10 MPa), emulating conditions of in situ reservoir environments, on the effectiveness of the biosurfactant producers was also assessed using a custom-designed high-pressure bioreactor. Within the examined temperature range, it was confirmed that B. subtilis produced the lipopeptide biosurfactant (surfactin) with and without oxygen using nitrate (NO3-) as the alternative electron acceptor. Thereby, the IFT was reduced from similar to 50 to similar to 10 mN/m and the wettability was modified from the values indicating an intermediate water-wet condition (theta = similar to 45-50 degrees) to a strong water-wet condition (theta = similar to 20-25 degrees). With the significantly improved capillary factor (gamma cos theta) by a factor of 4.4, the two-phase flow simulations using the pore network model estimated significant increases in oil recovery rates in microbially treated reservoirs. The lowest rate and amount of surfactin production were observed at 45 degrees C, suggesting that higher temperatures may not be favorable for surfactin production by Bacillus spp. These results provide unique quantitative experimental evidence corroborating the feasibility of utilizing biosurfactant-producing microorganisms for MEOR practices targeting reservoirs with high pressure and moderately high temperature.