Pesticides are transported into estuaries during rainfall events, where they may bioaccumulate and impact non-target organisms, including seafood species. Current-use pesticides (CUPs) have superseded legacy chlorinated pesticides due to lower predicted bioaccumulation potential, environmental persistence and human health impacts. Yet, little is known about the uptake and impacts of these chemicals on estuarine seafood species. Recently, concerns were raised by commercial fishers in New South Wales following several large-scale giant mud crab (Scylla serrata) mortality events, which fishers perceived were related to flooding-related stressors and contaminants, including pesticides. To validate these concerns, we assessed spatiotemporal trends in CUP accumulation by crabs in six estuaries spanning a gradient of anthropogenic land-uses and undertook a short-term ecotoxicology experiment. We identified 11 analytes accumulated in crab edible tissues, comprising various classes of insecticides, herbicides and fungicides, with CUP profiles varying among estuaries and collection periods, reflecting differences in catchment land uses and climatic conditions. Residue(s) were detected in ~60% of samples and although concentrations posed a negligible exposure-risk to seafood consumers, an ecological risk assessment identified that CUP-tissue concentrations in morbid crabs may have resulted in acute toxicity, aligning with fishers’ observations. We also experimentally tested the combined effects of propargite exposure (the most frequently detected compound) and salinity decline (i.e. a simulated rainfall runoff event) on crab physiology, flesh condition and behaviour, highlighting the value of sublethal biomarkers for detecting early responses to multiple stressors before effects become evident at higher levels of biological organisation. Collectively, these studies illustrate the value of fishers’ knowledge for informing targeted research on emerging contaminants and underscore the regulatory need for seafood surveillance and ecotoxicology testing on harvested species.