The Sambhar Salt Lake in Rajasthan, India, offers a unique opportunity to explore halophilic surfactin-producing microorganisms with industrial application. The phylogenetic study, comparative genomics combined with chemotaxonomic research provided insight into the interspecies relativeness of Bacillus sp. Considering the nonribosomal protein sequences (NRPS), Bacillus haynesii strain SAII strain and Bacillus swezeyi strain SEIII showed a high degree of conservation. In silico studies of the isolated Bacillus sp. confirmed the presence of NRPS indicating the presence of surfactin-type lipopeptide biosurfactants. Structural investigation using Fourier transform infrared spectroscopy, liquid quadrupole triple quadrupole mass spectrometry, 1 H Nuclear mass resonance of the crude biosurfactant revealed further similarities among these two Bacillus species. Methyl esters of the crude biosurfactant indicated high likeliness among them and indicated the presence of pentadecanoic acid and tetradecanoic acid as the crucial fatty acids. Enhanced recovery of oil from contaminated sand using 100% crude biosurfactant ranged from 20% AE 0.05 to 69.20% AE 1.22. This opens the door to a bright future for understanding the molecular diversity of closely related halophilic Bacillus sp. and the potential use of the extracted biosurfactants for bioremediation of contaminants.