Enzymatic and alkali pretreatments were employed to improve nickel biosorption capacity of Rhizomucor pusillus biomass. Pretreatment with 0.002–80 g l−1 NaOH and 0.0001–0.1 Anson Unit (AU) g−1 protease enhanced the biosorption capacity of fungal biomass. Increasing the concentration of NaOH from 0.002 to 5 g l−1 improved nickel removal from 93.2 to 100.0 % while untreated biomass showed 64.6 % Ni(II) removal. Pretreatment with higher concentrations of NaOH, 5–80 g l−1 resulted in nearly complete removal of nickel ions. Pretreatment of the biomass with 0.0001 AU g−1 protease improved the nickel removal to over 91 %, while increasing the enzyme loading to 0.1 AU g−1 improved the removal to 93 %. Untreated biomass removed 78.4, 63.0, and 96.3 % of chromium, copper, and lead ions, respectively, from a mixture solution of the ions. Respective metal removals were increased to 100, 98.9, and 100 % after pretreatment with 0.2 g l−1 NaOH solution and to 87.8, 86.7, and 100 % after the enzymatic pretreatment with 0.1 AU g−1 protease. Scanning electron microscopy analysis indicated that alkali and enzymatic pretreatments enhanced the porosity of the biomass. Furthermore, compositional analysis showed that both of the pretreatments removed a major part of fungal proteins (2.1–95.8 % removal). Glucosamine, N-acetyl glucosamine, and phosphates were the major ingredients of the pretreated biomass.