There is limited information about the factors that affect the power generation of single-chamber microbial fuel cells (MFCs) using soil organic matter as a fuel source. We examined the effect of soil and water depths, and temperature on the performance of soil MFCs with anode being embedded in the flooded soil and cathode in the overlaying water. Results showed that the MFC with 5 cm deep soil and 3 cm overlaying water exhibited the highest open circuit voltage of 562 mV and a power density of 0.72 mW m −2 . The ohmic resistance increased with more soil and water. The polarization resistance of cathode increased with more soil while that of anode increased with more water. During the 30 d operation, the cell voltage positively correlated with temperature and reached a maximum of 162 mV with a 500 Ω external load. After the operation, the bacterial 16S rRNA gene from the soil and anode was sequenced. The bacteria in the soil were more diverse than those adhere to the anode where the bacteria were mainly affiliated to Escherichia coli and Deltaproteobacteria. In summary, the two bacterial groups may generate electricity and the electrical properties were affected by temperature and the depth of soil and water.