Studies focused on limnology and quality of water bodies are essential to produce data that support the planning and management, diagnose the current situation and propose measures to minimize the possible negative impacts on water resources. The Tereza Botas creek drains the town of Poconé-MT, situated in the Pantanal border. In this sense, the objective of this study was to verify the variations in limnological characteristics along the longitudinal gradient and evaluate the water quality of the Tereza Botas watershed, identifying the impacts from anthropogenic activities of this important Pantanal watershed. Four sampling points along the longitudinal gradient of the water stream have been defined, and physical, chemical and microbiological variables were analyzed. The results indicated that the changes in limnological and water quality derive from the riparian vegetation removal, erosion and discharge of sewage in natura. In addition, high density of E. coli was observed in most of the sampled points, demonstrating that the lack of proper sanitation as well as the presence of rudimentary cesspit are negatively affecting water quality in the headwaters region.