The characteristics of four new proton-conducting membranes, Nafion112/polyaniline composite membranes of various compositions, are studied for application as membrane separators in microbial fuel cells. The composite membranes are made by immersing Nafion-112 membranes in a solution containing aniline for different immersion times. The presence of polyaniline and sulfonic functional groups in the composite membranes is confirmed by means of Fourier transform infrared analysis while their surface roughness is determined by using atomic force microscopy prior to microbial fuel cell operation. Biofouling on the membranes' surface is also examined by using a scanning electron microscope after microbial fuel cell operation. The polarization curves and, hence, the power density curves are measured by varying the load's resistance. The power density of the microbial fuel cell with the Nafion/polyaniline composite membranes improves significantly as the amount of polyaniline increases because the interaction between sulfonic groups in the Nafion matrix and polyaniline in the polyaniline domains increases proton conductivity. However, it declines after more polyaniline is added because of less conjugated bonding of polyaniline and sulfonic acid groups for larger polyaniline domains in the Nafion matrix. The voltage overpotential is also smaller as the amount of polyaniline increases. Biofouling also decreases with increasing polyaniline in the Nafion/polyaniline composite membranes because they have smoother surfaces than Nafion membranes. The results show that the maximum power generated by the microbial fuel cells with Nafion112-polyaniline composite membrane is 124.03 mV m−2 with a current density of 454.66 mA m−2, which is approximately more than ninefold higher than that of microbial fuel cells with neat Nafion-112. It can be concluded that the power density of the microbial fuel cell can be increased by modifying the Nafion membrane separators with more conductive polymers that are less susceptible to biofouling to improve its proton conductivity.