Abstract Single chamber MFC (SMFC) consisted of two separator-electrode assemblies (SEA) using low-pH distillery wastewater (DW) was operated under continuous mode. The electricity generation and microbial community were analyzed according to the external resistance (Rext; 0.1, 0.5, 1, and 5kΩ). The two SEAs exhibited different electricity generations, despite sharing the same anodic chamber. The SMFC showed the largest maximum power density (PDmax) of 3.7W/m3 (SEA 1) and 12.9W/m3 (SEA 2) at 5kΩ. These results demonstrated that low-pH wastewater could be sufficiently used as fuels for electricity generation. Pyrosequencing analysis showed that microbial communities at the phylum level were significantly different according to the Rext. The communities of SEA 1 were slightly different from those of SEA 2. In both SEAs, Firmicutes (>45%) were the most dominant at 0.1kΩ, while Firmicutes (>34%) and Caldiserica (>34%) were dominant at 5kΩ. Caldiserica sp. might significantly contribute to electricity generation under low-pH and high-Rext.