The largest economic, population, administrative, and service production of the State of Puebla (east-central Mexico) is concentrated in the Metropolitan Area of Puebla (MAP), and its effect on the water quality of the Atoyac River is substantial. The anthropogenic contamination of the Atoyac River and its tributaries in the MAP was evaluated and characterized. For this purpose, industry types and industrial density (ID) were identified, and the physical–chemical quality of water in the urban Atoyac, Rabanillo, Zapatero Rivers, and the Covadonga and Echeverría Dams were analyzed. In addition, the cytotoxicity of water was evaluated using the biomodel Allium cepa by analyzing the mitotic index (MI) and the interphase index (II). The correlation between the physical–chemical variables of water and MI was estimated. The results included 4500 industries, among which are the construction, metallurgy, metalworking, food, and textile industries. The highest ID was found in the municipality of San Pedro Cholula. The greatest anthropogenic impact occurred in the Rabanillo River and the Echeverría Dam. Throughout the watershed, anoxic conditions were registered and 18 chemicals, most of which are toxic and carcinogenic, were detected. A Correlation analysis showed that the greatest antimitotic effect in Allium cepa was induced by turbidity, chemical oxygen demand (COD), and lead (Pb), with correlation coefficients of −0.919, p = 0.008; −0.864, p = 0.013; and −0.692; p = 0.030, respectively. It was concluded that if the population, industry, and natural resources are associated in the MAP under current conditions, the outlook, if nothing changes, is that the degradation of the water resource will be disruptive and probably unsustainable for any type of use.