This study was aimed to determine the spatial variation in anthropogenic pressure exerted on surface water in the Podhale region (southern Poland), which is one of the most popular tourist destinations in Poland. The assessment was based on the dynamics and relationships between microbiological and chemical indicators of water quality throughout the major rivers of this region—Dunajec, Czarny Dunajec, Biały Dunajec, and Białka. Another aim was to assess the effect of land use on the quality of water in the studied rivers. The study was conducted over 1 year at 21 sampling sites distributed from the uppermost sections of rivers in the Tatra National Park through main tourist resorts until mouths of the considered rivers to the Czorsztyńskie Lake. Microbiological analysis comprised the prevalence of total and fecal types of coliforms and Escherichia coli, mesophilic, and psychrophilic bacteria. Chemical assays determined the concentrations of Na⁺, K⁺, NH₄ ⁺, Cl⁻, NO₃ ⁻, and PO₄ ³⁻. Temperature, electrical conductivity, and pH were measured onsite. It was demonstrated that there is a significant relationship between the predominant types of land use within individual catchments, which results in evident differences in the pollution of waters between the catchments. The results showed that increased share of built-up areas and arable land results in significant deterioration of water quality. Thus, waters of Czarny Dunajec were the cleanest, while Biały Dunajec was the most heavily contaminated. Also, spatial diversity in water quality was shown—the cleanest waters were sampled in the Tatra National Park and the pollution increased with the course of rivers. Point sources of pollution such as effluents from treatment plants or discharge of untreated sewage from households proved to be more important than non-point sources, such as surface runoff. Moreover, the important role of the Czorsztyńskie Lake in the purification of water was demonstrated.