Strain ZNI5, isolated from a hydrocarbon contaminated soil and identified as Bacillus subtilis after 16s rDNA sequence, grew and produced lipopeptides biosurfactants when cultured on glucose based media. After purification by anionic exchange chromatography and identification Reverse Phase High Performance Liquid Chromatography-Mass Spectrometry, the biosurfactant produced by ZNI5 were determined to be cyclic lipopeptides homologues. Four families of lipopeptides were identified by HPLC-MS analysis. They belongs to surfactin isoforms with molecular weights of 979, 993, 1007, 1021 and 1035 Da; iturin isoforms with molecular weights of 1028, 1042 and 1056 Da; Licheniformin with molecular weight of 1410 and newly identified isoforms named Inesfactin with molecular weights of 973 and 987 Da. Functional properties of the ZNI5 biosurfactant were studied. It was characterized as a powerful surface-active agent that decreases the surface tension of water from 72 mN/m to about 32 mN/m with a CMC value of 350 mg/L more efficient than chemical surfactants (Triton X100; CTAB and SDS). It has the capacity to disperse oil to about 80 mm at a concentration of 800 mg/L showing close efficiencies to the listed chemical surfactants. In addition, by studying the surface tension decrease capacity and the oil displacement activity, ZNI5 lipopeptide biosurfactant showed great thermal, pH and salts activity and stability enabling its use in the bioremediation fields and for diverse industrial applications.