Zooplankton are a key food source for juvenile fishes in estuaries worldwide, including California’s Sacramento–San Joaquin Delta (hereafter Delta); both zooplankton quality and quantity are critical to ecosystem health. Zooplankton may be affected by pesticides in water and the food web, and the Delta is known to contain complex pesticide mixtures. In this study, we evaluated pesticide concentrations in water and zooplankton in the northern Delta during (1) the summer–fall of 2017, 2018, and 2019, which included periods of augmented pulse flows from agriculture tailwater, and (2) across a full seasonal cycle from May 2019 to March 2020. We quantified changes in pesticide concentration in response to environmental factors. We found that zooplankton showed more frequent detections of hydrophobic pesticides compared to more frequent detections of hydrophilic compounds in water. Pesticide concentrations were influenced by flow, pesticide application, and season, but the effects of these environmental factors differed by habitat (Sacramento River or Yolo Bypass Toe Drain). Pesticides in water responded similarly to environmental factors in the Sacramento River and Yolo Bypass, whereas pesticides in zooplankton responded differently. In water, we found more detections and higher concentrations at higher flows in the Yolo Bypass and Sacramento River, but responses to pesticide application varied by habitat. Alternatively, pesticide concentrations in zooplankton increased in the Yolo Bypass with increasing flow (correlated with flow pulses) and changed seasonally; whereas, pesticide concentrations in zooplankton in the Sacramento River decreased at higher flows, and decreased with or did not respond to higher pesticide application in the watershed. Our study suggests that augmented flows—particularly those using agricultural tailwater—may have unintended negative ecological effects that could partially offset benefits to the food web and fishes in the northern Delta, underscoring the complex interplay among factors that drive increased pesticide exposure.