This study aims to optimize factors that affect power generation in microbial fuel cell (MFC) using a statistical experimental design. A MFC reactor with two compartments has been constructed from cheap materials and used for electricity generation. The performance of MFCs can be influenced by several factors, hence; the Plackett–Burman design was employed to determine the most significant factors that affect power generation. The factors that affected the electricity generation significantly were magnesium sulfate, yeast extract, and sodium acetate. A near optimum medium formulation was obtained using this method with increased power density by 71.57 %. In this respect, the three levels Box–Behnken design was applied. A polynomial model was created to correlate the relationship between the three significant variables and power density. Under the optimized condition, the power density was 390 mW/m². These results indicate that the optimized conditions accelerated the power generation and the maximum power point (MPP) was about 91.2 % higher than that recorded with the basal condition. Microbial fuel cells (MFCs) are new types of bioreactors that use bacteria to generate electricity from organic and nonorganic compounds and are considered one of the prospective alternative technologies.