Chesapeake Bay has been experiencing severe eutrophication-driven bottom hypoxia for several decades, yet the effects of hypoxia on its food webs, especially its pelagic components, remain largely enigmatic. To better understand how hypoxia influences the interaction and spatial overlap between planktivorous fishes (e.g., bay anchovy Anchoa mitchilli) and their mesozooplankton prey (e.g., Acartia tonsa), we contrasted the spatial distributions of these food-web components along the Bay's entire north–south axis during spring, summer, and fall 1996, 1997, and 2000, and along several cross-Bay transects during summer of these same years. Pelagic fish biomass was estimated with a surface-towed split-beam echosounder. Dissolved oxygen and mesozooplankton biomass were simultaneously quantified using a towed, undulating ScanFish sensor package. Results indicate that hypoxia can disrupt the diel vertical migration behavior of planktivorous fishes in Chesapeake Bay during summer by reducing access to bottom waters and forcing fish to reside in well-oxygenated surface or nearshore waters. In turn, reduced access to bottom waters reduces spatial overlap with mesozooplankton prey, which appear to use the hypoxic zone as a refuge. Ultimately, we discuss how these hypoxia-driven changes in behavior and spatial overlap may influence food web interactions and bay anchovy recruitment in Chesapeake Bay.