Surfactin is a lipopeptide, composed of one β-hydroxy fatty acid, a long fatty acid moiety, and seven amino acids. In this study, the biosurfactant biosynthesis genes; 4'-pantetheinyl transferase (sfp), phosphopantetheinyl transferase (sfpO), and surfactin synthetase (srfA) have been characterized from the marine sponge-associated Bacillus licheniformis NIOT-06 from the Andaman and Nicobar Islands. The purified recombinant biosurfactant revealed excellent emulsification activity with crude oil and kerosene. Reverse-phase high-performance liquid chromatography resolved the purified recombinant biosurfactant into several fractions and one of which had significant surface tension reducing property. Fourier transform infrared spectroscopy spectrum also revealed the presence of C-N-N, alkenes, and N-H as the functional groups, and a similar overlapping pattern was observed with that of standard lipopeptide surfactin. The diversity and phylogeny of sfp, sfpO, and srfA gene sequences were compared with other eubacteria. The sfp, sfpO, and srfA gene sequences obtained from Bacillus licheniformis NIOT-06 were diverse and appeared to be partially conserved when compared with the GenBank reported sequences of several eubacteria.