Microorganisms present in deep Atlantic coastal plain sediments affect the geochemical evolution of groundwater and its chemical and isotopic composition, yet the factors controlling their origin, distribution, and diversity are poorly understood. The evolution of the groundwater chemistry, the fractionation of stable carbon isotopes, and the groundwater age are all indicators of the inorganic and microbial reactions occurring along a given flow path from groundwater recharge to groundwater discharge. In this study, tritium, 14C, and groundwater chemistry along three flow paths of the Middendorf aquifer in South Carolina were analyzed. The 14C ranged from 89 percent modern carbon (pmC) in the recharge zone to 9.9 pmC in the distal borehole; the δ13C remained relatively constant at ∼−22‰, suggesting microbial oxidation of organic carbon. Carbon isotope analyses of particulate organic carbon from core sediments and groundwater chemistry were used to model the carbon chemistry; the groundwater ages obtained from 14C ranged from modern to 11,500 years B.P. The highest frequencies of occurrence, numbers, and diversity of aerobic and anaerobic bacteria were found in boreholes near the recharge zone where the calculated ages were <1000 years B.P. The transport of microorganisms from the recharge zone may be responsible for this distribution as well as the electron acceptors necessary to support this diverse community of bacteria. The presence of both aerobic heterotrophs and anaerobic sulfate- and iron-reducing bacteria in the core sediments suggested the occurrence of anaerobic microsites throughout this otherwise aerobic aquifer. The highest in situ microbial respiration rate, as determined by modeling, was found along a flow path near the recharge area. It is likely that the electron acceptors necessary for supporting a diverse microbial community are depleted by the time the groundwater residence time in the Middendorf aquifer exceeds several hundred years.