Biosurfactants are surface-active biomolecules with emulsifying properties. These versatile compounds have numerous applications across various industries and environmental contexts, including the remediation of areas contaminated by persistent pollutants such as hydrocarbons. This study focuses on the physicochemical characterization of a biosurfactant produced by a newly identified chrysene-degrading Achromobacter aegrifaciens strain. Biosurfactant production was evaluated using the emulsification index (E24). The research determined the biosurfactant's surface tension, critical micelle concentration (CMC), and stability under different temperatures, pH levels, and salt concentrations. The chemical components of the partially purified biosurfactant were analyzed using biochemical tests, thin layer chromatography (TLC), Fourier transform infrared (FTIR), nuclear magnetic resonance (HNMR) spectroscopy, and gas chromatography-mass spectrometry (GC-MS). After 96 h of incubation, A. aegrifaciens S5 yielded 1.68 g/L of biosurfactant, with a CMC of 50 mg/L. The isolated compound was identified as a novel glycolipopeptide biosurfactant, demonstrating high efficiency with an E24 of 88.4% and the ability to reduce the surface tension of water from 70.3 to 38 mN/m. The biosurfactant exhibited functional stability across temperatures from 4-80 °C, pH levels from 2 to 12, and salt concentrations of 1-10% while maintaining structural integrity up to 120 °C. Given these characteristics, the biosurfactant shows a promising potential for applications in petroleum, detergent, and food industries.