Rhamnolipids, produced during biodegradation, enhance the removal of hydrocarbons by decreasing the surface tension and increasing the bioavailability. Unlike the synthetic surfactants, rhamnolipids are very promising for bioremediation because of low toxicity and biodegradability. Therefore, the need for eco-friendly and biodegradable surfactants like rhamnolipids for reliable environmental cleanup is increasing. The present work revealed that kerosene degraders were the potential source of rhamnolipids. For this finding, six kerosene degrading bacterial isolates were investigated for their biosurfactant production. All of the isolates namely Pseudomonas plecoglossicida Ag10, Raoultella planticola Ag11, Staphylococcus aureus Ba01, Enterococcus faecalis Cr07, Acinetobacter johnsonii Sb01, and Pantoea agglomerans Sn11 were determined as rhamnolipid producers through oil spreading activity, emulsification index, and microbial adhesion to hydrocarbon tests. Blue agar plate method, thin layer chromatography, and Fourier transform infrared spectroscopy analyses were also performed to characterize the rhamnolipids. The results revealed that mono-rhamnolipids were dominant among kerosene degraders.