We investigated the mechanism of Congo red degradation and bacterial diversity in a single-chambered microbial fuel cell (MFC) incorporating a microfiltration membrane and air-cathode. The MFC was operated continuously for more than 4 months using a mixture of Congo red and glucose as fuel. We demonstrated that the Congo red azo bonds were reduced at the anode to form aromatic amines. This is consistent with the known mechanism of anaerobic biodegradation of azo dyes. The MFC developed a less dense biofilm at the anode in the presence of Congo red compared to its absence indicating that Congo red degradation negatively affected biofilm formation. Denaturing gradient gel electrophoresis and direct 16S ribosomal DNA gene nucleotide sequencing revealed that the microbial communities differed depending on whether Congo red was present in the MFC. Geobacter-like species known to generate electricity were detected in the presence or absence of Congo red. In contrast, Azospirillum, Methylobacterium, Rhodobacter, Desulfovibrio, Trichococcus, and Bacteroides species were only detected in its presence. These species were most likely responsible for degrading Congo red.