In this work, response surface optimization strategy was employed to enhance the biodegradation process of fresh palm oil mill effluent (POME) by Aspergillus niger and Trichoderma virens. A central composite design (CCD) combined with response surface methodology (RSM) were employed to study the effects of three independent variables: inoculum size (%), agitation rate (rpm) and temperature (°C) on the biodegradation processes and production of biosolids enriched with fungal biomass protein. The results achieved using A. niger were compared to those obtained using T. virens. The optimal conditions for the biodegradation processes in terms of total suspended solids (TSS), turbidity, chemical oxygen demand (COD), specific resistance to filtration (SRF) and production of biosolids enriched with fungal biomass protein in fresh POME treated with A. niger and T. virens have been predicted by multiple response optimization and verified experimentally at 19% (v/v) inoculum size, 100 rpm, 30.2°C and 5% (v/v) inoculum size, 100 rpm, 33.3°C respectively. As disclosed by ANOVA and response surface plots, the effects of inoculum size and agitation rate on fresh POME treatment process by both fungal strains were significant.