This study attempted to produce succinic acid from co-culture of Aspergillus niger, Trichoderma reesei, and Phanerochaete chrysosporium strains in a two-step fermentation: solid-state fermentation pre-culturing phase followed by slurry fermentation. Response surface methodology (RSM) via central composite design (CCD) was used to optimize and investigate the effects of slurry fermentation variables such as initial acetic acid concentration, initial pH, and solids loading on succinic acid yield. These fermentation variables were identified as key factors influencing succinic acid yield based on preliminary studies. Experimental validation of the optimum conditions (4.58 g/L of acetic acid, initial pH of 4.69, and 12.92% (w/w) of solids loading) produced 32.58 ± 0.18 g succinic acid per kilogram dry substrate, which agreed with the predicted yield of 32.25 g succinic acid per kilogram dry substrate. The interaction between the acetic acid and initial pH possibly affected the permeability of acetate and succinate anions to the cells resulting in the accumulation of extracellular succinic acid in the slurry. High solids loading was observed to limit mixing and mass transfer of the slurry components. The results of this study will be useful in performing kinetic studies to deepen the understanding of producing succinic acid using fungal co-culture.