AbstractNovel biosurfactant-producing strains were isolated from hydrocarbon-contaminated environments that exclusively utilize agro-waste as their primary carbon source for the expression of biosurfactants. These were quantified using various standardized methods. Among the agro-waste screened, Beta vulgaris (Beetroot) proved to be the most suitable substrate, for which the biosurfactants produced by three bacterial isolates–B. licheniformis STK01, B. subtilis STK02, and P. aeruginosa STK03–lowered the surface tension of the culture media to 30.0, 32.98, and 30.37 mN/m, respectively. The biosurfactants achieved considerable emulsification activity, particularly for heavy hydrocarbons, with the highest emulsification indices being 65.5% and 95% for anthracene and lubricant oil, respectively. The emulsion formed with lubricant oil was thermally stable even up to 50 °C for 21 days. The results showed the proficiency of the novel bacterial isolates used, as well as the suitability of solid agro-waste for biosurfactant production, thus suggesting that exclusive utilization of solid agro-waste is a promising option for use in biosurfactant production for environmental remediation. The outstanding emulsification activity and thermal stability demonstrated by the biosurfactants produced showed their potential applications in enhancing bioavailability and bioremediation of recalcitrant and hydrophobic environmental contaminants.