AIMS: The study of microbial inactivation rates in aquifer systems has most often been determined in aerobic and oxidized systems. This study examined the inactivation (i.e. loss of infectivity) of MS2, poliovirus type 1 (PV1) and Cryptosporidium parvum in an anaerobic and reduced groundwater system that has been identified as storage zones for aquifer storage and recovery (ASR) facilities. METHODS AND RESULTS: Anaerobic and reduced (ORP < (-) 250 mV) groundwater from an artesian well was diverted to an above-ground, flow-through mesocosm that contained diffusion chambers filled with MS2, PV1 or Cryptosporidium parvum. The respective infectivity assays were performed on microorganisms recovered from the diffusion chambers during 30- to 58-day experiments. The net reduction in infectivity was 5.73 log10 over 30 days for MS2, 5.00 log10 over 58 days for PV1 and 4.07 log10 over 37 days for C. parvum. The best fit inactivation model for PV1 was the log-linear model and the Weibull model for MS2 and C. parvum, with respective inactivation rates (95% confidence interval) of 0.19 (0.17-0.21) log10 day(-1) , 0.31 (0.19-0.89) log10 day(-1) and 0.20 (0.14-0.37) log10 day(-1) . CONCLUSIONS: The groundwater geochemical conditions in this aquifer enhanced the inactivation of MS2, PV1, and C. parvum at rates approximately 2.0-5.3-fold, 1.2-17.0-fold, and 4.5-5.6-fold greater, respectively, than those from published studies that used diffusion chambers in aerobic-to-anoxic groundwater systems, with positive redox potentials. SIGNIFICANCE AND IMPACT OF THE STUDY: Geochemical conditions like those in the aquifer zone in this study can naturally and significantly reduce concentrations of microbial indicators and pathogens of human health concern in injected surface water. Appropriate storage times for injected surface water could complement above-ground engineered processes for microorganism removal and inactivation (e.g. filtration, disinfection) by naturally increasing overall microorganism log-inactivation rates of ASR facilities.