Interests in biosurfactant in industrial and environmental applications have increased considerably in recent years, owing to their potential benefits over synthetic counterparts. The present study aimed at analyzing the stability and oil removal efficiency of a new lipopeptide biosurfactant produced by Paenibacillus sp. D9 and its feasibility of its use in biotechnological applications. Paenibacillus sp. D9 was evaluated for optimal growth conditions and improved production yield of lipopeptide biosurfactant with variations in different substrate parameters such as carbon (C), nitrogen (N), C:N: ratio, metal supplements, pH, and temperature. Enhanced biosurfactant production was observed when using diesel fuel and ammonium sulfate as carbon and nitrogen source respectively. The maximum biosurfactant yield of 4.11g/L by Paenibacillus sp. D9 occurred at a C/N ratio of 3:1, at pH 7.0, 30 degrees C, 4.0mM MgSO4, and 1.5% inoculum size. The D9 biosurfactant was found to retain surface-active properties under the extreme conditions such as high thermal, acidic, alkaline, and salt concentration. The ability to emulsify further emphasizes its potential usage in biotechnological application. Additionally, the lipopeptide biosurfactant exhibited good performance in the degradation of highly toxic substances when compared with chemical surfactant, which proposes its probable application in biodegradation, microbial-enhanced oil recovery or bioremediation. Furthermore, the biosurfactants were effective in a test to stimulate the solubilization of hydrophobic pollutants in both liquid environments removing 49.1 to 65.1% diesel fuel including hydrophobic pollutants. The study highlights the usefulness of optimization of culture parameters and their effects on biosurfactant production, high stability, improved desorption, and solubilization of hydrophobic pollutants.