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The highly toxic species of Chromium in its hexavalent state is an important hazard to the flora and fauna, causing a rupture in balance especially in aquatic environments. The removal of Cr(VI) ions from aqueous solutions using fungal biomass of Rhizopus sp. was investigated under batch experiments. The biomass was produced and treated with NaCl to compare pre-treated and untreated biosorbents capacity. Adsorption of Cr(VI) was investigated with a 23 experimental design to determine the best operational parameters including pH [2.0–4.0], temperature [20–40 °C] and agitation [50–150 rpm]. Maximum Cr(VI) uptake (99%) indicated that pH 2.0 is the optimal for Cr(VI) removal. Linear and non-linear kinetic models were evaluated. The best fitting for linear kinetics was the pseudo-second order linear equation and the Elovich model in its non-linear form, suggesting chemisorption as the controlling step of adsorption. Results followed Langmuir isotherm equation, the q m was 9.95 (mg·g−1) for Rhizopus sp. + NaCl. Thermodynamic parameters were calculated using the adsorption equilibrium constant obtained from Langmuir isotherm and indicated that the adsorption process was spontaneous and endothermic. The surface characteristics of the biomass were analyzed by Fourier transform infrared (FTIR) spectra; the analysis showed the involvement of amino groups in the bonding with Cr(VI). SEM and EDX analysis confirmed the presence of Cr in the biomass after adsorption. The results of these experiments may be utilized for modeling, simulation, and scale-up processes in the future.