Human activities threaten the integrity of watersheds. We aimed to investigate the impact of land use on water quality, adopting a multiscale approach. We collected water samples from twelve streams in Southern Brazil and conducted limnological analyses (physical, chemical, and biological) during the dry season. We used the water quality index based on the quality standards of Canada and Brazil. Land use percentage was measured in two groups (local scale and network scale). Environmental variables were summarized through Principal Component Analysis, and we organized them into Linear Models, integrating the percentage of land use classes and terrain slope in the Multifit formula. Statistical analyses were performed using the R software. Results indicated contamination by lead, chromium, copper, nitrogen, andEscherichia coliin water samples. The Canadian Water Quality Guidelines for the Protection of Aquatic Life resulted in an index ranging from 23.3 to 47.3, compared to the Brazilian Resolution No. 357/2005 for Class 2, which had an index ranging from 47.5 to 100. This disparity is attributed to the more rigorous and sensitive monitoring approach adopted by the Canadian guidelines. Riparian forests which are up to 50 m wide are associated with improved water quality. Agricultural and urban activities were the main contributors to water quality degradation in an area extending up to 1000 m from the watershed. We emphasize the importance of a multiscale approach in watershed management and public policies, considering not only riparian forest preservation, but also human activities throughout the watershed. It is crucial to prioritize science-based environmental public policies and strengthen enforcement to prevent increasingly pronounced environmental collapses. We have identified the urgency to reformulate CONAMA Resolution No. 357/2005 with a more conservationist and ecosystem-oriented approach, as well as to propose modifications to the Brazilian Forest Code, particularly regarding the buffer zones of permanent preservation areas. Thus, this study can provide insights, such as incorporating the “effect scale,” to enhance water resource management in landscapes heavily influenced by human action, contributing to the advancement of future research in freshwater ecosystems.