The Sado basin (~8000 km2) is an area where intensive agriculture occurs. However, this region still has few data about the water levels of priority pesticides such as fungicides, herbicides, and insecticides. Therefore, water samples were collected every two months at nine sites along the Sado River Estuary and analyzed by GC-MS/MS to determine the influx of pesticides in that ecosystem. More than 87% of the pesticides were quantified, and 42% and 72% were above the maximum totals established by the European Directives 98/83/EC and 2013/39/EU, respectively. Fungicides (91%), herbicides (87%), and insecticides (85%) attained average annual amounts of ≈3.2 µg/L, ≈1.0 µg/L, and ≈12.8 µg/L, respectively. A mathematical approach was used to evaluate the hazard of the pesticide mixture at the maximum concentrations found in this area. The assessment identified invertebrates as the most at-risk trophic level and identified two chemicals (chlorpyriphos and cyfluthrin) as the primary culprits. This assumption was supported by acute in vivo assays usingDaphnia magna. These observations, and the high concentrations of phosphates, indicate that the status of the Sado waters poses environmental and potential human health risks.