Protected areas (PAs) represent a powerful refuge for maintaining and safeguarding biodiversity. Generally, PAs are delineated to protect terrestrial taxa, providing incidental protection to the aquatic ecosystems within their borders. Here, we compare water quality within PAs and non-PAs in southern Brazil, encompassing remnants of the Atlantic Forest biome, to assess whether PAs serve as a buffer from external pressures for aquatic ecosystems within their boundaries. In addition to physicochemical and microbiological water parameters, we analysed 147 pesticide and 31 pharmaceutical compounds in water samples from 33 sites within and outside PAs. The water quality did not differ between PAs and non-PAs but indicated clear pollution from sewage discharges. We found 19 pesticides and five pharmaceuticals in streams within the study area. We detected pesticides in all sampling sites, with the herbicide 2,4-dichlorophenoxyacetic acid present in 91% of them. Our data show that PAs are insufficient means to mitigate the impacts stemming from their catchments, and the running water that reaches their domains already shows signs of anthropogenic interference, which may affect aquatic biodiversity. Protection and management measures require consideration of the whole watershed to protect freshwater habitats and biota.