In Latin America and the Caribbean, with a population of approximately 580 million inhabitants, less than 20 % of wastewater is treated. Megacities in this region face common challenges and problems related with water quality and sanitation, which require urgent actions, such as changes in the sustainable use of water resources. The Mexico City Metropolitan Area is one of the most populous urban agglomerations in the world, with over 20 million inhabitants, and is no exception to the challenges of sustainable water management. For more than 100 years, wastewater from Mexico City has been transported north to the Mezquital Valley, which is ranked as the largest wastewater-irrigated area in the world. In this study, bacteria and pathogenic protozoa were analyzed to determine the association between the presence of such microorganisms and water types (WTs) across sampling sites and seasons in Mexico City and the Mezquital Valley. Our results show a difference in microbiological water quality between sampling sites and WTs. There is no significant interaction between sampling sites and seasons in terms of bacterial concentration, demonstrating that water quality remains constant at each site regardless of whether it is the dry or the rainy season. The results illustrate the quantity of these microorganisms in wastewater, provide a current diagnosis of water quality across the area which could affect the health of residents in both Mexico City and the Mezquital Valley, and demonstrate the need to transition in the short term to treat wastewater from a local to a regional scale.