Volatile fatty acids (VFAs) are key intermediates in anaerobic digestion. Enriched acetogenic and methanogenic cultures were used for the syntrophic anaerobic digestion of VFAs in a continuous fixed-bed reactor at mesophilic conditions. The interactive effects of propionic (HPr), butyric (HBu), and acetic (HAc) acids were analyzed. Furthermore, hydraulic retention time (HRT) and methanogen-to-acetogen ratios (M/As) were investigated as the key microbiological and operating variables of VFA anaerobic degradations. Experiments were carried out based on central composite design (CCD) and results were analyzed using response surface methodology (RSM). Effluent concentrations of HPr, HBu, HAc, and biogas production rate (BPR) were directly measured as responses. The optimum conditions were found to be HPr = 1122.9 mg/L, HBu = 1792.4 mg/L, HAc = 1735.4 mg/L, HRT = 21 hours, and M/A = 2.4 (corresponding to the maximum VFA removal and BPR). The results of verification experiments and predicted values from fitted correlations were in close agreement at a 95% confidence interval.