Microorganisms indigenous to an oil reservoir were grown in media containing either sucrose or proteins in four steel vessels under anoxic conditions at 30 °C and 8.3 MPa for 30 days, to enrich biosurfactant producers. Fermentation of substrate was possible in the protein-containing medium and either fermentation or respiration through reduction of sulfate occurred in the sucrose-containing medium. Growth of microorganisms led to 3.4–5.4-fold surface tension reduction indicating production of biosurfactants in amounts sufficient for enhancement of gas-driven oil recovery. Analysis of sequenced cpn60 amplicons showed that Pseudomonas sp. highly similar to biosurfactant producing P. fluorescens and to Pseudomonas sp. strain TKP predominated, and a bacterium highly similar to biosurfactant producing Bacillus mojavensis was present in vessels. Analysis of 16S rDNA amplicons allowed only genus-level identification of these bacteria. Thus, cpn60-amplicon analysis was a more relevant tool for identification of putative biosurfactant producers than 16S rDNA-amplicon analysis.