Microbial fuel cells (MFCs), capable of concurrently treating sulfide and nitrate while generating electricity, were evaluated with various influent sulfide to nitrate molar ratios (S:N ratios). All MFCs displayed a good substrate removal capacity regardless of the S:N ratio (i.e., 5:0, 5:1, 5:2, and 5:3) and produced nitrogen gas, elemental sulfur, and sulfate as major products, while low S:N ratios mediated high sulfate conversion. By setting the S:N ratio to 5:2, the electricity generation was maximized, which was consistent with cyclic voltammetry (CV) curves. Through high-throughput sequencing, the taxonomic distribution of a microbial community in suspended sludge was analyzed; the diversity indices and a principal component analysis (PCA) suggest that the S:N ratios may affect the microbial community in MFCs. However, a Pearson correlation analysis infers that the S:N ratios do not have a significant impact on the richness and diversity of the microbial communities in MFCs involved in the simultaneous treatment of sulfide and nitrate (p > 0.05).