In Waorani communities of the Amazon basin of Ecuador, hepatitis B (HB) surface antigen was found in 14% of asymptomatic persons. The aim of this study was to identify the Waorani population in Ecuador and obtain an epidemiologically adequate coverage of vaccination against hepatitis B virus (HBV) among them. Between February and September 2010, three vaccination campaigns were completed as part of a collaborative activity between the Ecuadorian Ministry of Public Health and the community leaders in the intervention areas. In addition, in 10 of the 22 Waorani communities, during first contact, a questionnaire about risk factors for HBV infection was administered. A total of 16 Waorani communities were registered before the beginning of this intervention, but during the actual process, six additional communities were identified. In total, 1,251 community members were registered, 92.7% of which received all three doses of the vaccine. Among them, 83% of survey respondents reported having been vaccinated, but only 22% were able to show proof. Only 1% of respondents were intravenous drug users, whereas 22% had at least one tattoo. All self-identified as heterosexual, but only 32% reported using condoms during sexual intercourse. By March 2015, no new cases of HB were been reported in any of the Waorani communities. Herein, we report the importance of intersectoral collaboration to strengthen health interventions in an isolated population like the Waorani area.