Biosurfactant-producing microorganisms improve the efficacy of hydrocarbon biodegradation as the biosurfactant is essential in making hydrocarbons available for breakdown. The present study reports the isolation of biosurfactant-producing bacteria that can be used for crude oil remediation and to characterize the biosurfactant generated during the breakdown of crude oil. This study also reports evaluating the synergism and potentiality of biosurfactant-producing bacteria for simultaneous hydrocarbon biodegradation and power generation. Two bacterial strains (Bacillus subtilis strain B1 and Pseudomonas aeruginosa strain B2) were isolated from petroleum-contaminated soils, which are found effective in producing biosurfactants and degrading crude oil as the sole carbon source. B. subtilis B1 exhibited a higher potential for biosurfactant production and crude oil degradation than P. aeruginosa B2. The FTIR and GC-MS analysis were conducted for further characterization of the biosurfactant, which revealed that the surfactant produced by strain B1 and B2 was surfactin and rhamnolipid, respectively. The application of the B1 and B2 co-culture in microbial fuel cells (MFCs) showed synergism among them and resulted in a maximum power density production of 6.3 W/m3 with an open circuit voltage of 970 mV while degrading 2.5% v/v crude oil containing anolyte. The findings indicate that the co-culture of isolated crude oil-degrading strains has great potential for enhanced power generation and the bioremediation of hydrocarbon-contaminated environments. Moreover, the synergism of isolated strains in MFCs suggested their potent applicability in environmental, energy, and industrial sectors as an economical and feasible alternative to the existing technologies.