Resumo. AIM: This study evaluated the impacts of anthropogenic activities upstream of conservation areas on the Paraibuna river and its implications for freshwater biodiversity. METHODS: The study was carried out in two units, Cunha and Santa Virginia, of the Serra do Mar State Park (SP), located in the Atlantic Rain Forest. Five sampling sites were defined, four along the Paraibuna river and one in the Ipiranga river, the latter fully inserted into the protected area. Physical, chemical, microbiological and ecotoxicological data were obtained from surface water as well as aquatic macroinvertebrates. RESULTS: The results showed that the waters of the Paraibuna river have low anthropogenic interference. However, conductivity, turbidity, coliforms, iron, total phosphorus and nitrate showed a gradient improving its water quality from upstream to downstream, indicating the existence of erosion and introduction of organic debris in the basin. The BMWP index, varying from 58 to 190, also showed the good condition of the river to aquatic biota, with predominant Excellent quality diagnosis. The values of this index and the richness index (S) outlined a similar gradient but with the lowest values recorded in P3. CONCLUSIONS: The results showed that the upstream activities alter the natural condition of the Paraibuna river and its biota and that the protected areas provides environmental services reducing these impacts. The ideal situation in order to ensure the conservation of the freshwater biota of the Paraibuna river would be the incorporation of parts of the upstream area into the protected area and convert occupied areas into Sustainable Use Area, that guarantee the adoption of sustainable techniques to the existing land uses and the application of aquatic life protection indicators for monitoring the water quality of the river.