Due to an ever-increasing population and developments in technology, the demand for energy has been increasing daily. In order to meet this demand, renewable and alternative energy sources that do not harm the environment are often recommended. One of these alternative sources can be obtained from human wastewater and is known as the Microbial Fuel Cell (MFC). This study aimed to estimate the electricity production performance of MFC, using Nafion 117 membrane with 10 × 10 and 11 × 11 cm2 area by both active and sediment sludges. The responses obtained from 16 physical experiments performed according to the L16(24 × 22) Taguchi orthogonal index were analyzed by the response surface method (RSM). According to the analysis, it was determined that the quadratic polynomial equations created for the estimation of the reaction values of the active and sediment sludge had validity over 90%. The ANOVA analysis applied to determine parameter ethicalities also showed that the most effective parameter on the reactions from the sludge types considered was the resistance at different rates. Thus, the results suggest that the orthogonal design-based RSM model is an effective tool that provides key information for optimizing energy harvesting from MFC technology and saves time concerning experimental work.