Biosurfactants are surface-active natural polymers produced within several microorganisms and are secreted outside the cellular environment. They are the focus of modern researches due to their eco-friendly nature and high production capability using low-cost agro-industrial wastes. In this research, we have evaluated Aspergillus niger (A. niger) for its biosurfactant production potential during solid state fermentation of banana stalks powder. The native strain of A. niger produced 2.3 g/L of biosurfactant with 49.74 cm² oil displacement, 57% emulsification index and an emulsification activity of 1.024 (OD₅₄₀). Sequential mutagenesis was induced in the native strain of A. niger by exposing the strain with different concentrations of ethidium bromide (EtBr), for different time periods. Significantly higher amount of biosurfactant (3.3 g/L) was obtained from the mutant strain A. niger M2 exposed to 50 µg/10 mL of EtBr for 60 min. The screening tests revealed the improvement in oil displacement (59.81 cm²), emulsification index (62.3%) and emulsification activity (OD₅₄₀, 1.262) of biosurfactant. FTIR analysis confirmed the presence of amine, amide, fatty acids and triglycerides functional groups. The maximum biosurfactant synthesizing mutant (A. niger M2) was further optimized using RSM under CCD. After optimization, the highest biosurfactant (5.50 g/L) was obtained at 35 °C temperature, 7 pH, 5.75 g substrate concentration and 168 h of overall incubation period. In conclusion, the cost-effective production of biosurfactant, along with novel structural and multifunctional characteristics, this study may be useful for different industrial and biotechnological applications.