Aquifer storage and recovery (ASR) can augment water supplies and hydrologic flows under varying climatic conditions. However, imposing drinking water regulations on ASR practices, including pre-treatment before injection into the aquifer, remains arguable. Microbial inactivation data—Escherichia coli, Pseudomonas aeruginosa, poliovirus type 1 and Cryptosporidium parvum—were used in a human health risk assessment to identify how the storage time of recharged water in the Floridan Aquifer enhances pathogen inactivation, thereby mitigating the human health risks associated with ingestion. We used a quantitative microbial risk assessment to evaluate the risks for a gastrointestinal infection (GI) and the associated disability-adjusted life years (DALYs) per person per year. The risk of developing a GI infection for drinking water no longer exceeded the suggested annual risk threshold (1 × 10−4) by days 31, 1, 52 and 80 for each pathogen, respectively. DALYs per person per year no longer exceeded the World Health Organization threshold (1 × 10−6) by days 27, <1, 43 and 72. In summary, storage time in the aquifer yields a significant reduction in health risk. The findings emphasize that considering microbial inactivation, caused by storage time and geochemical conditions within ASR storage zones, is critical for recharge water treatment processes.