Bioelectricity production from microbial fuel cell (MFC), fed with synthetic wastewater can achieve both wastewater treatment and energy production. Two different types of separators were used in this study to compare their performance in an air cathode MFC. Power generation and substrate utilization from the wastewater was compared for these membranes. The maximum power density obtained with Zirfon® was 424.50 mW/m2 and of 16.98 W/m3 (volumetric) while with Fumasep® was 38.03 mW/m2 and 1.52 W/m3 (volumetric). The internal resistance obtained with Zirfon® increased from initial 0.38 Ω cm2 to 1.72 Ω cm2 while with Fumasep® increased from 2.57 Ω cm2 to 10.92 Ω cm−2 at the end of the experiment. The results showed that the MFC with Zirfon® stabilized early and the anodic and cathodic cell potentials reached maximum earlier as compared to Fumasep®. Impedance spectra confirmed the lower resistance of the Zirfon® as compared to Fumasep® and this may be the reason for its better performance. Zirfon® might be a good option for the future scale up of MFCs because of its low cost and low internal resistance in comparison to other membranes.