Microbial fuel cells (MFCs) can convert chemical energy to electricity using microbes as catalysts and a variety of organic wastewaters as substrates. However, electron loss occurs when fermentable substrates are used because fermentation bacteria and methanogens are involved in electron flow from the substrates to electricity. In this study, MFCs using glucose (G-MFC), propionate (P-MFC), butyrate (B-MFC), acetate (A-MFC), and a mix (M-MFC, glucose:propionate:butyrate:acetate = 1:1:1:1) were operated in batch mode. The metabolites and microbial communities were analyzed. The current was the largest electron sink in M-, G-, B-, and A-MFCs; the initial chemical oxygen demands (CODini) involved in current production were 60.1% for M-MFC, 52.7% for G-MFC, 56.1% for B-MFC, and 68.3% for A-MFC. Most of the glucose was converted to propionate (40.6% of CODini) and acetate (21.4% of CODini) through lactate (80.3% of CODini) and butyrate (6.1% of CODini). However, an unknown source (62.0% of CODini) and the current (34.5% of CODini) were the largest and second-largest electron sinks in P-MFC. Methane gas was only detected at levels of more than 10% in G- and M-MFCs, meaning that electrochemically active bacteria (EAB) could out-compete acetoclastic methanogens. The microbial communities were different for fermentable and non-fermentable substrate-fed MFCs. Probably, bacteria related to Lactococcus spp. found in G-MFCs with fermentable substrates would be involved in both fermentation and electricity generation. Acinetobacter-like species, and Rhodobacter-like species detected in all the MFCs would be involved in oxidation of organic compounds and electricity generation.