Seafloor massive sulfides are a potential energy source for the support of chemosynthetic ecosystems in dark, deep-sea environments; however, little is known about microbial communities in these ecosystems, especially below the seafloor. In the present study, we performed culture-independent molecular analyses of sub-seafloor sulfide samples collected in the Southern Mariana Trough by drilling. The depth for the samples ranged from 0.52 m to 2.67 m below the seafloor. A combination of 16S rRNA and functional gene analyses suggested the presence of chemoautotrophs, sulfur-oxidizers, sulfate-reducers, iron-oxidizers and iron-reducers. In addition, mineralogical and thermodynamic analyses are consistent with chemosynthetic microbial communities sustained by sulfide minerals below the seafloor. Although distinct bacterial community compositions were found among the sub-seafloor sulfide samples and hydrothermally inactive sulfide chimneys on the seafloor collected from various areas, we also found common bacterial members at species level including the sulfur-oxidizers and sulfate-reducers, suggesting that the common members are widely distributed within massive sulfide deposits on and below the seafloor and play a key role in the ecosystem function.