Volcanic ocean crust contains a global chemosynthetic microbial ecosystem that impacts ocean productivity, seawater chemistry, and geochemical cycling. We examined the mineralogical effect on community structure in the aquifer ecosystem by using a four-year in situ colonization experiment with igneous minerals and glasses in IODP Hole 1301A on the Juan de Fuca Ridge. Microbial community analysis and scanning electron microscopy revealed that olivine phases and iron-bearing minerals bore communities that were distinct from iron-poor phases. Communities were dominated by Archaeoglobaceae, Clostridia, Thermosipho, Desulforudis, and OP1 lineages. Our results suggest that mineralogy determines microbial composition in the subseafloor aquifer ecosystem.