This objective of this study is to conduct a systematic investigation of the effects of configurations, electrolyte solutions, and electrode materials on the performance of microbial fuel cells (MFC). A comparison of voltage generation, power density, and acclimation period of electrogenic bacteria was performed for a variety of MFCs. In terms of MFC configuration, membrane-less two-chamber MFCs (ML-2CMFC) had lower internal resistance, shorter acclimation period, and higher voltage generation than the conventional two-chamber MFCs (2CMFC). In terms of anode solutions (as electron donors), the two-chamber MFCs fed with anaerobic treated wastewater (AF-2CMFCs) had the power density 19 times as the two-chamber MFCs fed with acetate (NO(3)(-)2CMFCs). In terms of cathode solutions (as electron acceptors), AF-2CMFCs with ferricyanide had higher voltage generation than that of ML-2CMFCs with nitrate (NO(3)(-)ML-2CMFCs). In terms of electrode materials, ML-2CMFCs with granular-activated carbon as the electrode (GAC-ML-2CMFCs) had a power density 2.5 times as ML-2CMFCs with carbon cloth as the electrode. GAC-ML-2CMFCs had the highest columbic efficiency and power output among all the MFCs tested, indicating that the high surface area of GAC facilitate the biofilm formation, accelerate the degradation of organic substrates, and improve power generation.