Single-chamber microbial fuel cells (MFCs) acclimated with glucose, butyrate, propionate, acetate, and a mixture of the four were operated with brewery wastewater (BWW) under a fed-batch mode. Glucose-fed MFC (GW-MFC) showed the highest maximum power density (PDmax) of 1,519 mW/m2, followed in order by acetate-fed MFC (AW-MFC), mixed substrates-fed MFC (MW-MFC), butyrate-fed MFC (BW-MFC), and propionate-fed MFC (PW-MFC). After changing to BWW, power production was decreased for all MFCs. MFC acclimated with glucose showed the highest PDmax of 890 ± 12 mW/m2, followed in order by MW-MFC, AW-MFC, BW-MFC, and PW-MFC. The PDmax in BWW-MFC, which was acclimated and operated with BWW, of 552 mW/m2 was less than that of GW-MFC and MW-MFC but more than that of AW-MFC, BW-MFC, and PW-MFC. MFCs with fermentable substrates were less affected by the BWW. Gammaproteobacteria, including Pseudomonas sp., Acinetobacter sp. and Xanthomonas axonopodis, were found in all MFCs with pure substrates and Rubrivivax benzoatilyticus, thiobacillus sp. and Denitratisoma oestradiolicum belonging to Betaproteobacteria were newly detected in all MFCs when the substrate was changed to BWW.