The Atoyac River crosses the metropolitan area of Puebla-Tlaxcala in Mexico and presents a state of pollution that has been scarcely studied. In 2016, the water quality of the river was evaluated based on physicochemical and bacteriological parameters, under the guidelines established for the Maximum Permissible Limit (MPL) for Aquatic Life Protection (ALP) and Agricultural Irrigation (AI). The sampling sites were the Covadonga Dam, the Echeverría Dam, and an irrigation canal. Water from wells and a spring in the Emilio Portes Gil (EPG) population that uses Atoyac water for agricultural irrigation was also analyzed. The data obtained from the river were compared with the 2011 data published in the declaration of classification of Atoyac and Xochiac or Hueyapan and its tributaries. There was a notable increase in hemical oxygen demand (COD) (49%) and in heavy metals with varying percentages. The anoxic condition of the river (mean 1.47 mg of O₂/L) with large populations of coliform bacteria was demonstrated, 11 pathogenic members of the Enterobacteriaceae were found, and high organic pollution concentrations were shown, particularly during droughts. Irrigation and well water was contaminated with fecal bacteria (104–549 NMP/100 mL), which included pathogens.