ABSTRACT Head-starting of the federally endangered Houston toad ( Anaxyrus houstonensis ), that is, the release of egg strands, tadpoles, and metamorphic juveniles produced in captivity into the original breeding ponds, requires assessment of potential threats for the transmission of pathogens from captive to free-ranging toads. We used Illumina-based 16S rRNA V3 amplicon sequencing to investigate the community structure of bacteria from skin lesions of captive Houston toad and habitat (pond) samples. Proteobacteria, alone or together with Actinobacteria and, in some samples, Cyanobacteria represented virtually all reads in tissue lesion samples, whereas pond samples were much more diverse, with Acidobacteria, Actinobacteria, Bacteriodetes, Chloroflexi, Cyanobacteria, Firmicutes, Planctomycetes, Proteobacteria, and Verrucomicrobia present with little variation between samples. If present in lesions, Actinobacteria were largely represented by Mycobacteriaceae, and here mainly by one sequence identical to sequences of members of the Mycobacterium chelonae – abscessus complex. In pond samples, mycobacteria represented only a small portion of the actinobacteria, although at higher diversity with six distinct reads. Sequences for reads obtained from pond samples were identical to those representing the M. chelonae – abscessus complex, a group with Mycobacterium marinum , Mycobacterium kansasii , Mycobacterium avium , a group with Mycobacterium vaccae , Mycobacterium fortuitum , Mycobacterium poriferae , and a group with Mycobacterium elephantis and Mycobacterium celeriflavum , whereas sequences of high similarity were detected for reads related to those of Mycobacterium holsaticum , Mycobacterium pallens, and Mycobacterium obuense , and Mycobacterium goodii . Our results indicated that lesions observed on the Houston toad in captivity are not the result of mycobacteria in every case, and that the presence of mycobacteria in the captive colony does not represent a novel pathogen threat to the wild populations because such bacteria are also seen in the natural pond habitats for the Houston toad.