Considering the projected demand for protein supplementation in animal feed, as well as prioritizing plant-based protein provision for the growing human population, great stress is imposed on conventional protein sources, calling for new sustainable alternatives. In this regard, the production and application of single-cell proteins (SCPs) has proven to be a promising alternative. Therefore, in this study, volatile fatty acids (VFAs) effluents recovered from anaerobically digested FW, CKM, CM, and their combinations were applied for the cultivation of edible filamentous fungi Aspergillus oryzae. The biomass was further evaluated considering its protein, fat and alkali insoluble material contents. The maximum fungal biomass yielded of 0.47 ± 0.00 and 0.37 ± 0.00 g dry biomass/g tVFAsCODeq.consumed, with up to 47% protein and 5% fat content successfully cultivated in shake flasks and bench scale reactors, respectively. In addition to the production of protein-rich biomass, significant reductions in medium COD (25–58%) and ammonium (33–48%) were achieved. The results presented in this research work imply that using waste-derived VFAs for the production of animal feed grade SCP is an innovative approach that can contribute to the economy and sustainability of animal feed production process.