Rivers in Southeast Brazil are essential as sources of drinking water, energy production, irrigation, and industrial processes. The Piracicaba, Capivari, and Jundiaí rivers basin, known as the PCJ basin, comprises major cities, industrial hubs, and large agricultural areas, which have impacted the water quality in the region. Emerging contaminants such as pesticides, hormones, pharmaceuticals, industrial chemicals, and per- and polyfluoroalkyl substances (PFAS) are likely to be released into the rivers in the PCJ basin; however, the current Brazilian legislation does not require monitoring of most of these chemicals. Thus, the extent of emerging contaminants pollution and their risks to aquatic and human life in the basin are largely unknown. In this study, we investigated the occurrence of several pesticides, hormones, pharmaceuticals, and personal care products in 15 sampling points across the PCJ basin, while industrial chemicals and PFAS were assessed in 11 sampling points. The results show that agriculture and industrial activities are indeed causing the pollution of most rivers. Multivariate analysis indicates that some sampling points, such as Jundiaí, Capivari, and Piracicaba rivers, are largely impacted by pesticides used in agriculture. In addition, to the best of our knowledge, this is the first study reporting the presence of PFAS in rivers in São Paulo, the most populous state in Brazil. Four out of eight species of PFAS assessed in our study were detected in at least 5 sampling points at concentrations ranging from 2.0 to 50.0 ng L−1. The preliminary risk assessment indicates that various pesticides, caffeine, industrial chemicals, and PFAS were present at concentrations that could threaten aquatic life. Notably, risk quotients of 414, 340, and 178 were obtained for diuron, atrazine, and imidacloprid, respectively, in the Jundiaí River. Our study suggests that establishing a comprehensive monitoring program is needed to ensure the protection of aquatic life and human health.