The maximum performance of microbial fuel cells (MFCs) is significantly affected by the reduction reactions in the cathode, but their optimum condition is not fully understood yet. The air-cathode MFC operations with different separators (Nafion 117 and polypropylene (PP80) were evaluated at various relative humidity (RH) at the cathode chamber. Air cathode MFCs with a Nafion 117 separator at RH of 90 ± 2% produced the highest cell voltage of 0.35 V (600 Ω) and power density of 116 mW/m2. With a PP80 separator, the maximum power generation of 381 mW/m2 was obtained at a relatively lower RH of 30 ± 2%. The cyclic voltammogram and Tafel analysis indicated that the best performance of cathodic oxygen reduction reactions could be observed at 90% RH for Nafion and 50% RH for the PP80 separator. Additionally, the RH conditions also affected the anodic reactions and oxygen mass transfer rates to the anode chamber through the cathode and separators. This study suggests that the optimum RH condition at the cathode is important in order to obtain a high performance of MFC operations and needs to be controlled at different optimum levels depending on the characteristics of the separators.