Oil palm empty fruit bunch (OPEFB) was biologically pretreated using Pleurotus floridanus LIPIMC996 supplemented with various concentrations of manganese and nitrogen and incubated for 35 days at 30°C, and enzyme activities of manganese peroxidase (MnP) and laccase were examined. When OPEFB was supplemented with manganese, the highest lignin reduction was achieved up to 25.0±5.6% at the addition of 200 μg Mn2+/g OPEFB.In addition, Pleurotus floridanus LIPIMC996 grew best on OPEFB supplemented with 800 μg Mn2+/g OPEFB. When OPEFB was supplemented with nitrogen, the highest lignin reduction was achieved up to 27.2± 3.5% at the addition of 20 mM nitrogen. The best growth of Pleurotus floridanus was also achieved with the addition of 20 mM nitrogen. The addition of nitrogen and manganese on OPEFB did not significantly affect the activity of MnP and laccase.