The use of surfactin, a powerful biosurfactant, is generally hampered by poor production yield. Consequently, identification of new producers and the study of operational parameters are essential. We identify Bacillus sp. H2O‐1 as Bacillus velezensis, a species previously not investigated for its biosurfactant production. Among the nitrogen sources we tested, (NH4)2SO4 and NH4NO3 were the most appropriate for surfactin production, reaching 608.5 and 659.5 mg L−1, respectively. Only temperature affected the production, whereas rotation and the C/N ratio did not. Biosurfactants can be used in enhanced oil recovery (EOR) in reservoirs located in the presalt and postsalt layers, where conditions of temperature, pressure, and salinity are quite varied, requiring a study of the stability of these molecules under these conditions. We found the surfactin produced by B. velezensis to be stable at different temperatures, pH, and ionic strengths. We evaluated the concurrent effects of different salinity, temperature, and pressure conditions on surface and interfacial activities of this surfactin. Overall, we found the surfactin produced by B. velezensis H2O‐1 to have considerable potential for industrial applications, mainly due to the stability of its physical and chemical characteristics when subjected to different temperatures, pressures, and salinities, in addition to its low toxicity.