The sources of microbial and chemical contamination of groundwater are numerous and have severe implications for public health. The objective was to determine the microbiological and physicochemical quality of wells in the west and east of the Cuautla-Yautepec aquifer in Mexico. Wells showed bacteriological contamination in at least one sampling. Coliform values were lower than the maximum permissible limit indicated in the Mexican Ecological Criteria of Water Quality (1000 colony forming unit (CFU)/100 ml of fecal coliforms) for supply sources. The number of isolated amoebae was low, but these were present all year round. Amoebae were found in 71.7% of the samples and belonged to 13 genera. The most frequent amoeba, Hartmannella, occurring in 44% of the samples, has been associated with eye and brain infection, but its role as a cause of infection has not been confirmed. A gradient was observed for dissolved solids according to altitude; the concentrations of dissolved solids increased in wells with lower altitudes. Total hardness values were above 180 mg/L CaCO3, therefore the water is considered very hard, and both carbonate and non-carbonate hardness was detected. The average values of physicochemical parameters were below the maximum permissible limits indicated in the Mexican official norm.