Objective: The present work was undertaken to isolate an efficient biosurfactant-producing bacterium, optimize its production under different environmental and nutritional conditions, and characterize the structural and functional properties of the biosurfactant for prospective industrial use. Methods: Environmental samples from soil and wastewater were subjected to preliminary screening through hemolytic activity, oil displacement, and emulsification index (E24) assays. The most promising isolate was identified by morphological and biochemical tests, followed by molecular confirmation using 16S rRNA gene sequencing. Production parameters were optimized by varying carbon and nitrogen sources, culture pH, temperature, and incubation period. Crude biosurfactant was recovered through acid precipitation and solvent extraction. Its functional attributes were examined through surface tension reduction, critical micelle concentration (CMC) determination, emulsification potential, and stability across pH, temperature, and salinity gradients. Structural features were analyzed by Fourier-transform infrared spectroscopy (FTIR) and gas chromatography– mass spectrometry. Results: The identified Bacillus subtilis strain effectively reduced surface tension from 72.0 to 28.6 mN/m and exhibited a high emulsification index (75.4±1.2% with kerosene). Optimal production occurred with glucose (2% w/v) and ammonium nitrate (0.5% w/v) at pH 7 and 30°C after 72 h, yielding 4.85±0.15 g/L of crude biosurfactant. The CMC was estimated at 35 mg/L. The compound retained stability between pH 4–10, at temperatures up to 100°C, and in salinity up to 10% NaCl. FTIR and GC–MS confirmed a glycolipid nature, composed mainly of long-chain fatty acids and sugar moieties. Conclusion: The biosurfactant derived from B. subtilis displayed strong surface-active properties, appreciable yield, and robustness under diverse physicochemical conditions. These features underline its promise for applications in petroleum recovery, bioremediation, effluent treatment, and biopharmaceutical formulations, underscoring its potential as a safe and sustainable alternative to synthetic surfactants. © 2025 The Authors.