Cr(VI) tolerance in Aspergillus flavus, strain SFL, isolated from tannery effluent was measured and compared with a reference strain of A. flavus, A1120. On solid medium, SFL had a high level of Cr(VI) tolerance (1,600 mg/L), which was 16 times that of A1120 and greater than most previously analyzed fungal strains. When in 100 mg/L of Cr(VI), SFL completely depleted Cr(VI) within 72 h while A1120 depleted 85% of Cr(VI). SFL was more effective in reducing extracellular Cr(VI) than A1120. While A1120 showed greater biosorption of Cr(VI) than SFL, intracellular accumulation was approximately 50% greater in SFL and was more energy‐dependent than A1120. Cr(VI) modified the external surface of the hyphae. Cr speciation detected the presence of only Cr(III), corresponding to Cr(OH)3, which precipitated on the hyphal surface. Cr(VI) bound to the functional groups carboxyl, amine, and hydroxyl in both SFL and A1120. Transmission electron microscopy energy‐dispersive X‐ray detected Cr on the fungal wall and within membrane‐bound organelles of the cytoplasm. In conclusion, the greater tolerance of SFL to Cr(VI) relative to A1120 is due to more effective energy‐dependant uptake of Cr(VI) into the cell and increased capacity of SFL to store Cr in intracellular vacuoles compared with A1120.