A microbial electrosynthesis cell (MES) is an electrochemical technique in which electrolithoautotrophic electroactive microbes fix carbon dioxide (CO2) to longer-chain volatile fatty acids (VFAs). The synthesis of VFAs from CO2 requires optimization to improve the MES performance for industrial feasibility. This work studied the effect of different parameters, such as pH, headspace gas pressure, ethanol concentration, electrolyte, and trace element concentrations, on VFA synthesis from CO2 that used mixed anaerobic consortia in serum bottles. The operational parameters were varied according to a central composite design (CCD) and response surface methodology (RSM). A global optimum for the response variables was determined. The optimum values of different operating factors to maximize VFA production that was obtained from the optimizations were 1.12 × 105 Pa pressure, pH 7.149, ethanol = 2,318.7 mg/L, three times the standard Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) 300 electrolyte concentration and five times the standard DSMZ 300 trace elements concentration. The experimental study was validated in triplicate, which considered the optimal values. The rate of total VFA production that was obtained from the operational parameters experimental study was 43.7 ± 5.9 mg/L/day under this optimized condition, which agreed with the predicted value of 30.39 mg/L/day. For the media optimization, validation of the experimental study was conducted at the optimal values, and their experimental response was 79.75 ± 19.26 mg/L/day, and the predicted response was 75.89 mg/L/day for total VFA production rates. © 2023 American Society of Civil Engineers.