Hypoxia and acidification are commonly coupled in eutrophic aquatic environments because aerobic respiration is usually dominant in bottom waters and can lower dissolved oxygen (DO) and pH simultaneously. However, the degree of coupling, which can be weakened by non-aerobic respiration and CaCO3 cycling, has not been adequately assessed. In this study, we applied a box model to 20 years of water quality monitoring data to explore the relationship between hypoxia and acidification along the mainstem of Chesapeake Bay. In the early summer, dissolved inorganic carbon (DIC) production in mid-bay bottom waters was dominated by aerobic respiration, contributing to DO and pH declines. In contrast, late-summer DIC production was higher than that expected from aerobic respiration, suggesting potential buffering processes, such as calcium carbonate dissolution, which would elevate pH in hypoxic waters. These findings are consistent with contrasting seasonal relationships between riverine nitrogen (N) loads and hypoxic and acidified volumes. The N loads were associated with increased hypoxic and acidified volumes in June, but only increased hypoxic volumes in August, when acidified volume declines instead. Our study reveals that the magnitude of this decoupling varies interannually with watershed nutrient inputs, which has implications for the management of co-stressors in estuarine systems.