The present study deals with the development and application of a proton-exchange polymer membrane separator consisting of graphene oxide (GO), poly(vinyl alcohol) (PVA), and silicotungstic acid (STA) in a single-chambered microbial fuel cell (sMFC). GO and the prepared membranes were characterized by FT-IR spectroscopy, XRD, SEM, TEM, and AC impedance analysis. Higher power was achieved with a 0.5 wt % GO-incorporated PVA-STA-GO membrane compared to a Nafion 117 membrane. The effects of oxygen crossover and membrane-cathode-assembly (MCA) area were evaluated in terms of current density and Coulombic efficiency. The electrochemical behavior of the membrane in an MFC was improved by adding different amounts of GO to the membrane to reduce biofouling and also to enhance proton conductivity. A maximum power density of 1.9 W/m(3) was obtained when acetate wastewater was treated in an sMFC equipped with a PVA-STA-GO-based MCA. Therefore, PVA-STA-GO could be utilized as an efficient and inexpensive separator for sMFCs.