In the last years, issues related to intensive agriculture have been found in protected areas potentially harming wildlife. This study aimed to analyze a wide range of pesticides in water and sediments of two protected areas namely Doñana Natural Park (DNP) and Tablas de Daimiel National Park (TDNP) performing an environmental risk assessment in order to highlight potential risks to living organisms derived from pesticide burden. Higher pesticide load was found in DNP than TDNP with similar distribution profiles, with pyrethroid insecticides (PYRs) the main detected class. Particularly problematic are two PYRs, cyhalothrin and fenvalerate, which were detected at high concentrations that can pose a high risk to aquatic organisms. In addition, despite being detected at lower concentrations, the presence of chlorpyrifos, cypermethrin, and permethrin in water, and of chlorpyrifos, dicofol, and diflufenican in sediments, must be taken into account due to their potential risks for aquatic organisms. Moreover, some banned pesticides such as dimethoate, terbutryn, diazinon, and tricyclazol were detected in water at levels which deserve further investigation to assess their potential sources, including potential illegal practices.