Knowledge is limited on the spread of bacteria from genus Chlamydia in Brazil. This study included a sero-epidemiological survey of 2,086 samples from native Indian populations of the Brazilian Amazon region. Sera were screened using indirect immunofluorescence assay for detection of antibodies to C. trachomatis serotype L2, followed by microimmunofluorescence assay using fifteen C. trachomatis and C. pneumoniae serotypes as antigen substrates. Antibody prevalence was 48.6%, but there was a large prevalence range among the groups, including those that had never been challenged with the bacteria, as well as those in which almost all individuals had been infected. Titration of IgG antibodies and detection of specific IgM in high-titer samples showed the persistence of Chlamydia in 6.1% of the reactive individuals, who probably play an important role as reservoirs for dissemination of the bacteria. Specific seroreactivity to C. trachomatis showed the presence of serotypes A, B, Ba, D, E, G, H, I, and L1 in the geographic area surveyed. Furthermore, the survey showed that C. pneumoniae was also infecting these individuals. Both species may be involved in a significant human disease burden that merits further clarification.