The main factors conditioning nickel biosorption by non-living fungal biomass of Pleurotus mutilus were studied by the means of two experimental approaches. The surface of fungal biomass was characterized by pHZPC determination, Boehm titration and FTIR spectroscopy. The results demonstrated that the pHZPC is found to be at pH = 7.94, and the number of acidic sites (4.33 meq g−1) is higher than the number of basic sites (0.58 meq g−1). Without the chemical precipitation of Ni(OH)2, the one variable at time (OVAT) approach illustrates at its equilibrium time equal to 79 min, that the maximum nickel biosorption was achieved at pH 8 and optimal concentration of 488 mg L−1. However, the particle size does not affect nickel biosorption. The comparison of the optimal conditions obtained from OVAT and Taguchi methods shows comparable values of pH and equilibrium time, while differences in Ni(II) concentration and particle size were observed. Furthermore, the combination of the four factors at their optimum level improves the biosorption capacity compared to OVAT method (enhancement of 36.23%). The Taguchi method revealed that the contribution of the controlled factors on Ni(II) biosorption followed the following order: Ni(II) (76.98%) > pH (18.16%) > time (3.20%) > particle size (1.66%). The combination of the variation factors could enhance the biosorption capacities of Pleurotus mutilus biomass for use in practical applications.