Background: Biosurfactants are surfactants derived from several types of microorganisms such as bacteria, yeasts and fungi as membrane components or secondary metabolites. Objective: To improve the output of biosurfactants as a low-toxic, biocompatible alternative for chemical surfactants utilized in a wide range of applications in today's industry. Materials and Methods: Different culture media were used inisolation of the biosurfactant (BS) producers. By using 16S rRNA, the most effective bacterial isolate was examined. For the optimization circumstances, Plackett Burman and Box-Behnken designs were employed. Purification and characterization of biosurfactant was done. Results: Only one isolate (BS) out of 63 isolates was selected for its potential ability of biosurfactant production, which lowered the surface tension (38.1 mN/m) and exhibited excellent emulsifying index against benzene (60.92 %) as well as oil spreading activity (9.76 cm), which was analyzed as Bacillus altitudinis AHMNAZ2 with accession number OP807875.1. The highest biosurfactant production yield was 2570 mg/L. The biosurfactant was lipopeptide in origin, identified as an iturin. Isolated iturin can be employed as an antibacterial agent against various Gram +ve and Gram -ve bacteria as well as some fungi. It showed remarkable action against the cell lines of breast cancer (MCV- 7) and colon cancer (HCT- 116). Conclusion: The investigation found a cheaper way of synthesis of biosurfactants from industrial wastes, delivering a twofold benefit of reducing pollution of environment and manufacturing useful biotechnological biosurfactant products, in addition to biosurfactant synthesis with high potency.