In this study, we have established a monitoring scheme to determine the presence and distribution of widely used pharmaceuticals, pesticides, organophosphate esters (OPEs), and perfluoroalkyl substances (PFAS) in water bodies from Important Bird and Biodiversity Areas (IBAs) from Spain. The monitoring scheme included the georeferenced sampling of rocky mountains, Atlantic forest, riparian forest, Mediterranean forest, agricultural, inland aquatic and coastal aquatic IBAs, with the aim to evaluate the impact of widely used chemicals in those aquatic resources. Water samples were extracted using a generic solid-phase extraction protocol and analyzed by 3 analytical methods based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Quality parameters such as compound recovery, intra and inter-day variation, linearity and limits of detection were calculated in order to validate the methods. In addition, the ionization conditions and the optimization of the most appropriate transitions permitted unequivocal identification. Once the sampling and analytical procedure was set up, 59 target compounds were monitored in 63 samples. Pharmaceuticals, followed by pesticides, OPEs, and PFAS were widespread along all IBAs studied at concentrations from 0.5 to 41083 ng/L. Overall, this study highlights the need to monitor the presence of contaminants in areas of high ecological interest to contribute to pollution control and mitigation toward the protection of biodiversity.