On July 25, 2016, turbid water and dead corals, sponges and other invertebrates were discovered at the East Bank (EB) of the Flower Garden Banks (FGB) National Marine Sanctuary. Mortality was spread over 0.06 km2, with up to 80% coral mortality reported in some areas. Within days, response efforts were underway to investigate the potential mechanisms leading to the mortality event. Hydrographic surveys, moored buoy data, and a regional hydrodynamic model were used to characterize water chemistry, hydrography, and microbial communities within the FGB. Low salinity (~ 31–33), total alkalinity (~ 2284–2330 µmol kg−1), and dissolved inorganic carbon (DIC, ~ 1968–2011 µmol kg−1) were detected in surface waters over the EB and eastern stations, revealing the presence of river-derived water. The Mississippi/Atchafalaya rivers were the primary sources of freshwater during the event, although Texas rivers, all of which had unusually high discharge during 2016, contributed approximately one-fifth to the total freshwater mass. At 75 m depth, high density, salinity, DIC, ammonium, and abundance of microbial taxa associated with deep waters were coincident with low temperature and aragonite saturation state at the northern and eastern stations, indicating a deeper source water at these stations. Cross-slope density gradients were also consistent with an upwelling circulation pattern. Using these observations and data, we hypothesize that the mortality event was most likely caused by the combination of two processes. The turbid freshwater layer inhibited photosynthesis, leading to net respiration of coral reef organic matter. Additionally, deep, dense waters upwelled onto the bank and formed a stratified bottom layer, which prevented re-oxygenation from the overlying water column and led to localized areas of hypoxia within pockets on the reef. Hypoxia likely formed rapidly, within two days. Moving forward, high-frequency temporal measurements of oxygen and carbonate chemistry are critical for monitoring risks (e.g., hypoxia and acidification) associated with freshwater discharge and upwelling, since these processes may adversely affect coral reef health.