Water plays a fundamental role in preserving life and is recognized as a human right; however, population growth has become a challenge to face regarding water quality and the health of aquatic ecosystems. The Guandu River Basin is an important source of water for the Metropolitan Region of the State of Rio de Janeiro, but it receives a large supply of untreated effluent, due to the urban influence, a study is necessary to assess the presence of bacteria that may be pathogenic, increasing the risk of disease. Metagenomics is a promising analysis technique, allowing us to understand microbial diversity in complex environments, such as aquatic ecosystems, enabling the identification of resistant microorganisms and the understanding of their resistance mechanisms. The concentration of resistomes found shows the importance and continued need for studies and monitoring in the Guandu River to better understand how they affect water quality and health.