Citrinin, a nephrotoxic mycotoxin produced by several fungal strains belonging to the genera Penicillium, Aspergillus, and Monascus. Generally found in stored grains and after their harvest. The objective of the present investigation was to study the antimicrobial activity (anti-fungal) of aqueous leaf extract of Aerva lanata and to optimize its conditions for the maximum inhibition of citrinin and fungal biomass by Aspergillus niger. Optimization of culture conditions was carried out using Box-Behnken method of response surface methodology. Extent of inhibition of citrinin was carried out using HPLC and reduction in fungal biomass was carried out using dry cell weight after comparing with controls. Optimized culture conditions as per the point prediction tool were found to be 11.27 mg/L for concentration of Aerva lanata extract, nine and half days of incubation period and temperature of 25.5 °C resulted in maximum inhibition of citrinin. These optimized values of tested parameters were and compared with control citrinin production (243.28 mg/L) and dry cell weight production (362.28mg).An average of 87.77±1.21% inhibition of citrinin and 80.02±1.42% of dry cell weight was obtained in an optimized medium at 9.5th d of fermentation with 97.82 % and 96.21% validity, respectively.