The effect of operating modes on the simultaneous sulfide and nitrate removal were studied in two-chamber microbial fuel cells (MFCs). The batch and continuous operating modes were compared and evaluated in terms of substrate removal and electricity generation. Upon gradual increase in the influent sulfide concentration from 60 to 1,020 S mg L(-1), and the hydraulic retention time decrease from 17.2 to 6 h, the MFC accomplished a good substrate removal efficiency whereby nitrogen and sulfate were the main end products. The removal efficiency of the MFC in the continuous mode was much higher than that in the batch mode, and its current densities in the continuous mode were more stable and higher than in the batch mode, which could be explained by the linear relationship between electrons released by the substrates and accepted on the electrodes. The electricity output in the continuous mode of the MFC was higher than that in the batch mode. MFC's operation in the continuous mode was a better strategy for the simultaneous treatment of sulfide and nitrate.