Widespread lead (Pb) contamination prompts various environmental problems and accounts for about 1% of the global disease burden. Thus, it has necessitated the demand for eco-friendly clean-up approaches. Fungi provide a novel and highly promising approach for the remediation of Pb-containing wastewater. The current study examined the mycoremediation capability of a white rot fungus,P. opuntiae, that showed effective tolerance to increasing concentrations of Pb up to 200 mg L−1, evidenced by the Tolerance Index (TI) of 0.76. In an aqueous medium, the highest removal rate (99.08%) was recorded at 200 mg L−1whereas intracellular bioaccumulation also contributed to the uptake of Pb in significant amounts with a maximum of 24.59 mg g−1. SEM was performed to characterize the mycelium, suggesting changes in the surface morphology after exposure to high Pb concentrations. LIBS indicated a gradual change in the intensity of some elements after exposure to Pb stress. FTIR spectra displayed many functional groups including amides, sulfhydryl, carboxyl, and hydroxyl groups on the cell walls that led to binding sites for Pb and indicated the involvement of these groups in biosorption. XRD analysis unveiled a mechanism of biotransformation by forming a mineral complex as PbS from Pb ion. Further, Pb fostered the level of proline and MDA at a maximum relative to the control, and their concentration reached 1.07 µmol g−1and 8.77 nmol g−1, respectively. High Pb concentration results in oxidative damage by increasing the production of ROS. Therefore, the antioxidant enzyme system provides a central role in the elimination of active oxygen. The enzymes, namely SOD, POD, CAT, and GSH, served as most responsive to clear away ROS and lower the stress. The results of this study suggested that the presence of Pb caused no visible adverse symptoms inP. opuntiae. Moreover, biosorption and bioaccumulation are two essential approaches involved in Pb removal byP. opuntiaeand are established as worthwhile agents for the remediation of Pb from the environment.