<p>Fungal isolates were obtained from local drainage systems and agricultural fields, showing diverse morphologies and pigmentation. Among them, one isolate (AN_1116) exhibited strong tolerance to manganese (Mn²⁺) and chromium Cr(VI) stress and was identified as <i>Aspergillus niger</i> through morphological and molecular characterization. Growth assays revealed concentration-dependent inhibition, with tolerance up to 800&#xa0;mg/L for Mn²⁺ and 600&#xa0;mg/L for Cr(VI). The biosorption potential of <i>A. niger</i> was optimized using response surface methodology (RSM), considering biomass concentration, pH, and contact time. Experimental biosorption assays achieved maximum removal efficiencies of 91% for Mn²⁺ and 85.72% for Cr(VI), demonstrating the high metal uptake capacity of the fungal biomass. Numerical optimization using the desirability function predicted optimal Mn²⁺ removal at 4.46&#xa0;g/L biomass, pH 4.35, and 56.37&#xa0;min contact time, yielding a predicted efficiency of 71.42%, while optimal Cr(VI) removal was predicted at 3.04&#xa0;g/L biomass, pH 6.59, and 130.99&#xa0;min, with a predicted efficiency of 70.64%. Structural characterization revealed enhanced surface porosity after NaOH pretreatment, while FTIR analysis confirmed the involvement of carboxyl, hydroxyl, and amino functional groups in metal binding. Adsorption isotherm modeling showed that the Freundlich model best described the process, suggesting multilayer adsorption on heterogeneous surfaces. Phytotoxicity tests on maize, wheat, barley, and sorghum showed 25–40% higher germination and root growth in treated wastewater than in untreated samples, confirming reduced toxicity. Overall, <i>A. niger</i> biomass demonstrates strong potential as an eco-friendly and cost-effective biosorbent for Mn²⁺ and Cr(VI) removal from contaminated wastewater, supporting sustainable agriculture and environmental management.</p>