Delignification is critical in the production of biofuel from a potentially abundant renewable resource, lignocellulosic waste. One of the classes of lignolytic enzymes are peroxidases. Manganese sulphate (Mn2+) has a significant impact on the peroxidase activity of lignolytic fungi. Along with induction, the suppression of peroxidase activity by Mn2+ has been observed. Peroxidase regulation is governed by three critical Mn2+ concentrations: the minimum inductive concentration (MIC), the peak concentration (PC) and the minimum suppressive concentration (MSC). The induction and suppression of enzyme activity were not associated with fungal growth capacity, but with a specific enzyme response to the nutritional conditions. Manipulation of the carbon and nitrogen sources shifted the peroxidase suppression by Mn2+ to high concentrations and, hence, increased the peroxidase tolerance to Mn2+ and, consequently, peak peroxidase activities. The manipulation of carbon and nitrogen sources allowed increasing the peak concentrations of Mn2+ and corresponding peroxidase activity up to 0.5 mg/mL and 180.70 AU (173% increase compared to standard) in C. subvermispora, and up to 0.05 mg/mL and 151.23 AU in D. squalens (91% increase compared to standard). Thus, manipulation of carbon and nitrogen sources is a useful tool in enhancing fungi peroxidase tolerance to Mn2+ and improving the delignification of lignocellulosic biomass by fungi to prepare an easily consumable substrate for economically viable biofuel production.