Waterborne pathogens, associated with poverty and poor sanitary conditions, are a major cause of morbidity and mortality worldwide. There are limited data on the epidemiology of waterborne pathogens in indigenous populations living in the Amazon region. We did a cross-sectional survey in two indigenous Shuar communities in the Amazon region of Ecuador in which we documented the presence of pathogens representing different sources of environmental contamination of water. We detected protozoa and soil-transmitted helminths by microscopy of fecal samples and the presence of IgG antibodies to hepatitis A and Leptospira spp. in blood samples from individuals older than 2 years and collected data by questionnaire on sociodemographic factors and knowledge of infectious diseases. Seroprevalence for hepatitis A and Leptospira spp. were 98.1% (95% CI: 97.0-99.8) and 50.0% (95% CI: 43.3-56.6), respectively, whereas 62.6% (95% CI: 55.8-69.4) had enteric parasites in stool samples. In participants older than 6 years, eight of 10 had evidence of infection with or exposure to at least one of the pathogens studied. Although prevalence of pathogens varied by age, it did not vary significantly by gender, temporal migration, illiteracy, perceived morbidity, receipt of conditional cash transfers, water boiling practices, poor housing conditions, and anthropometric status. These findings indicate a high level of contamination of drinking water by human pathogens in these indigenous communities and the need for interventions to improve access to and use of clean drinking water in these marginalized communities.