The study aimed to assess the occurrence and the environmental risk of a group of 51 selected pesticides in the Guadiana Basin (a biodiversity hotspot, in the Mediterranean). The most abundant pesticides were bentazone and 2,4-D, while terbuthylazine together with terbutryn constituted the most ubiquitous pesticides. Eighteen out of the 38 pesticides detected are no longer approved in Europe, and 5 of them are included in the list of priority substances. The risk assessment showed that azinphos ethyl, diflufenican, irganol, imidacloprid, and oxadiazon occurred occasionally, but always in concentrations above their respective ecotoxicological threshold value. Contrary, bentazone, terbuthylazine, and terbutryn presented a high risk in most of the sampled locations and periods.The site-specific risk assessment showed a spatial and temporal pattern, with a higher risk occurring mainly in intermittent streams, in the drought period. The presence of pesticides banned from the EU market since 2009 showed the importance of improving the monitoring process, to identify the main sources of pollution and the fate of these emerging compounds. The results showed the need of implementing actions to improve the sustainable use of pesticides in agricultural areas, working with farmers and management entities to reduce the contamination of aquatic ecosystems. Transboundary water governance is also required to solve potential transboundary contamination problems.