Microbial electrosynthesis (MES) systems can convert CO₂ to acetate and other value-added chemicals using electricity as the reducing power. Several electrochemically active redox mediators can enhance interfacial electron transport between bacteria and the electrode in MES systems. In this study, different redox mediators, such as neutral red (NR), 2-hydroxy-1,4-naphthoquinone (HNQ), and hydroquinone (HQ), were compared to facilitate an MES-based CO₂ reduction reaction on the cathode. The mediators, NR and HNQ, improved acetate production from CO₂ (165 mM and 161 mM, respectively) compared to the control (without a mediator = 149 mM), whereas HQ showed lower acetate production (115 mM). On the other hand, when mediators were used, the electron and carbon recovery efficiency decreased because of the presence of bioelectrochemical reduction pathways other than acetate production. Cyclic voltammetry of an MES with such mediators revealed CO₂ reduction to acetate on the cathode surface. These results suggest that the addition of mediators to MES can improve CO₂ conversion to acetate with further optimization in an operating strategy of electrosynthesis processes.