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Biosurfactants are one of the microbial bioproducts that are in most demand from microbial-enhanced oil recovery (MEOR). We isolated and screened potential biosurfactant-producing bacteria, followed by biosurfactant production and characterization, and a simulation of the MEOR application to biosurfactants in a sand-packed column. Isolate screening was conducted based on qualitative (hemolytic blood assay and oil-spreading test) and semi-qualitative (emulsification assay and interfacial tension measurement) parameters. Bacterial identification was performed using 16S rRNA phylogenetic analysis. Sequential isolation yielded 32 bacterial isolates, where Pseudomonas sp. G3 was able to produce the most biosurfactant. Pseudomonas sp. G3 had the highest emulsification activity (Ei = 72.90%) in light crude oil and could reduce the interfacial tension between oil and water from 12.6 to 9.7 dyne/cm with an effective critical-micelle concentration of 0.73 g/L. The Fourier transform infrared spectrum revealed that the biosurfactant produced was a glycolipid compound. A stable emulsion of crude extract and biosurfactant formed at pH 2–12, up to 100 °C, and with a NaCl concentration of up to 10% (w/v) in the response-surface method, based on the Box–Behnken design model. The sand-packed column experiment with biosurfactant resulted in 20% additional oil recovery. Therefore, this bacterium and its biosurfactant show potential and the bacterium is suitable for use in MEOR applications.