Subsurface sediments obtained from three cores drilled to depths of 260 m below the surface in South Carolina were analyzed for heterotrophic bacteria; N2‐fixing microaerophiles; and nitrifying, sulfur‐oxidizing, and H2‐oxidizing lithotrophic bacteria. In addition, pore waters were extracted for chemical analysis of inorganic nitrogen species, sulfate, dissolved organic carbon, pH, and Eh. Autotroph populations were generally less than 103most probable number (MPN) g‐1dry sediment with sulfur‐oxidizing bacteria, detected in 60% of the sediment samples, being the most frequently encountered group. Nitrifying bacteria were detected mainly in sediments from one borehole (P28), and their populations in those sediments were correlated with pore‐water ammonium concentrations. Populations of heterotrophic bacteria in 60% of the sediments were greater than 106colony forming units (CFU) g‐1dry sediment and were typically lower in sediments of high clay content and low pH. Microaerophilic N2‐fixing bacteria were cultured from >50% and bacteria capable of growth on H2were cultured from 35% of the subsurface sediments examined. Sediment texture, which controls porosity, water potential, and hydraulic conductivity, appears to be a major factor influencing microbial populations in coastal plain subsurface sediments.