Oxygen depletion in coastal waters is increasing globally due primarily to eutrophication and warming. Hypoxia responses to nutrient loading and climate change have been extensively studied in large systems like the Chesapeake Bay and the Baltic Sea, while fewer studies have investigated smaller, shallower hypoxic zones. Thus, an improved understanding of the interactions of eutrophication and warming on hypoxia expansion (or reduction) in the wide variety of different estuarine environments is needed. We examined interannual controls on oxygen depletion in the Patuxent River estuary, a eutrophic sub-estuary of Chesapeake Bay where seasonal hypoxia develops annually. We conducted a spatial and temporal analysis of dissolved oxygen (DO) trends, timing, and several metrics of depletion over a long-term record (1985–2021). We found an internally generated hypoxic zone that initiates in the middle estuary, spreading upstream and downstream as the summer progresses, and that hypoxic volume days (HVD) have been increasing (0.11 per year, p = 0.03) over the record despite reduced watershed nitrogen loads and stable phosphorus loads. River flow and temperature have been increasing and are major drivers of increased HVD, with river flow explaining 40% of the interannual variation in HVD (temperature has increased 0.03 and 0.06 °C per year in summer and fall, respectively). Apparent oxygen utilization (AOU) is increasing in bottom waters in the fall, consistent with increasing trends of both water temperature and stratification strength. HVD was negatively related (r2 = 0.34, slope = −0.59*HVD) to the biomass of benthic invertebrates in the middle region of the estuary, suggesting that benthic forage for higher trophic levels will be limited by sustained hypoxia. These results indicate that current and future climate variability plays an important role in regulating oxygen depletion in the Patuxent River estuary, which reinforces the need to factor climate change into strategies for the restoration and management of estuaries.