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Groundwater is heavily exploited for a variety of uses. Depending on their structure, the wells from which water is extracted can act as an entry point/gateway for a variety of microbiological contaminants, which can cause numerous adverse health effects. This study aimed to identify the microorganisms present in the groundwater in the Western Amazonian city of Porto Velho, using a methodology that can be deployed in other city centers. We collected 74 water samples from both dug and drilled wells in March, August and November 2018. Total coliforms were detected in 96% of dug wells and 74% of drilled wells. Thermotolerant coliforms were found in 90% of dug wells and 61% of drilled wells. Biochemical identification indicated 15 genera of bacteria. The genera Escherichia, Enterobacter, Cronobacter and Citrobacter had the highest prevalence. The genera Pseudomonas and Enterococcus were also detected. Thermotolerant coliforms showed higher values when the water flow was higher. Our results indicate high fecal contamination and higher susceptibility to contaminants in shallow wells compared to deep wells. These findings reflect the precariousness of WASH (water, sanitation and hygiene) services and the importance of effective actions to combat groundwater degradation, improve the quality of the environment, and protect public health.