|Abstract: Biosurfactants (BS) are amphiphilic molecules from microbial secondary metabolism capable of reducing surface and interfacial tension between non-miscible phases, thus having a wide range of industrial and environmental applications. In this context, the present work aimed to study the biosurfactant produced by the bacterial strain Ar70C7-2, isolated from rocky samples of a deep offshore Brazilian oil reservoir, to evaluate its application in the oil industry. Its optimized production conditions, chemical structure, stability in extreme conditions, surface-active properties in porous media, and antibacterial action were evaluated. Strain Ar70C7-2 showed 100% similarity to Bacillus alveayuensis and was able to produce 0.27 g/L of crude BS in the Mineral Medium with glycerin and NH4NO3 at a ratio of 2, under fixed conditions of 70 g/L NaCl, incubation at 55 °C, and no stirring. The semi-purified BS presented emulsification index (E24) above 55%, surface tension of 31.8 mN/m, interfacial tension (against n-hexadecane) of 17.6 mN/m and CMC of 65 mg/L. The BS belongs to the class of lipopeptides, with a protein composition of 7% and fatty acid chain ranging from C14 to C18, with homologue C16 as the most abundant. Mass spectra of BS indicate the presence of surfactin, iturin, and fengycin families. Semi-purified BS was able to remove approximately 30% of the motor oil (10W40) present in oil-soaked sand, as tested by the contaminated sand washing method; it also promoted the alteration of carbonate wettability, as demonstrated in the flotation test and by the Washburn method. BS inhibited growth of the sulfate-reducing bacteria (SRB) Desulfovibrio marinus BRS-1 at concentrations of 2250 µg/mL and showed bactericidal action at concentrations of 2500 µg/mL. These results demonstrate that the biosurfactant produced by strain Ar70C7-2 has the potential for application in MEOR, bioremediation, and controlling SRB growth, encouraging further studies to validate its prospect in laboratory and field conditions.