We surveyed the carbonate system along the main channel of the Chesapeake Bay in June 2016 to elucidate carbonate dynamics and the associated sources of oxygen-consuming organic matter. Using a two endmember mixing calculation, chemical proxies, and stoichiometry, we demonstrated that in early summer, dissolved inorganic carbon (DIC) dynamics were controlled by aerobic respiration in the water column (43%), sulfate reduction in the sediment (39%), atmospheric CO₂ invasion (13%), and CaCO₃ dissolution (5%). A mass balance of the DIC concentration and its stable isotope suggested that the apparent δ¹³C of oxygen-consuming organic matter was −19.4 ± 0.3‰. The bulk composition of particulate organic matter also reflected a dominance of algal material (C/N = ~ 6, δ¹³C > −25‰). Therefore, we concluded that the decomposition of autochthonous organic matter (i.e., eutrophication-stimulated primary production) was the dominant process consuming oxygen, while allochthonous organic matter (terrestrially derived) made minor contributions to oxygen consumption in the hypoxic zone in June 2016. These findings in the Chesapeake Bay contrast with another hypoxic estuarine ecosystem, the Pearl River Estuary in China where allochthonous organic matter contributed significantly to oxygen consumption. The differences between these two systems in terms of hydrology, quantity and quality of organic matter, and physical characteristics are discussed to yield new insights on the formation and maintenance of hypoxia. In both systems, autochthonous organic matter dominates oxygen depletion, indicating that nutrient management and reduction are useful actions to control and mitigate the occurrence of hypoxia for the restoration of ecosystem.