Marine oil pollution is a persistent environmental hazard that necessitates eco-compatible remediation strategies. This study reports the isolation and identification of biosurfactant-producing bacterial strains from oil-contaminated seawater collected from Ennore Harbor, Chennai, India. A total of 21 morphologically distinct isolates were obtained and evaluated for their biosurfactant-producing abilities using CTAB (Cetyl Tri Ammonium Bromide) agar, hemolysis, drop collapse, oil spreading, and emulsification index (E24) assays. Among the isolates, Bacillus cereus Is15 exhibited the highest and most consistent biosurfactant activity. Molecular identification via 16S rRNA sequencing revealed Is15 as Bacillus cereus Is15. Optimization studies indicated that peptone and glucose, at pH 7 and 37 °C, significantly enhanced the biosurfactant yield (0.120 g) and crude oil degradation (1.084 g). GC-MS (Gas Chromatography-Mass Spectrometry) analysis indicated the presence of compounds with surface-active properties, including isopropyl myristate and tetradecanoic acid. The application of the biosurfactant in a laboratory-scale oil degradation assay mimicking marine conditions resulted in a substantial reduction in total petroleum hydrocarbons (1.186 g), comparable to that of sodium dodecyl sulfate (SDS; 1.512 g). These findings highlight the potential of B. cereus Is15-derived biosurfactants as efficient and environmentally sustainable agents for marine oil bioremediation.