The effect of initial acetic acid concentration on succinic acid production from biologically pretreated birchwood chip-soybean hull mixture using co-culture of Aspergillus niger, Phanerochaete chrysosporium, and Trichoderma reesei in a slurry batch fermentation set-up was investigated. Acetic acid was supplied in various initial concentration: 1.65 g/L, 3.75 g/L, and 5.85 g/L. Low initial acetic acid resulted in decline of succinic acid production which coincided with declining acetic acid concentration. On the other hand, both medium and high initial acetic acid sustained higher succinic acid production with nearly constant acetic acid concentration throughout the five-day slurry batch fermentation. The residual sugar concentration was also observed as this was indirectly affected by the presence of acetic acid in the mixture. The presence of acetic acid in the fermentation media plays a crucial role in the production of succinic acid from a non-conventional source, the fungal co-culture.