A health assessment of the captive island population of Guam rail (Gallirallus owstoni) included a review of pathology records, diet analysis, and physical examination and diagnostic findings in prerelease and captive breeding populations (n  =  100 and n  =  55, respectively). The general health of domestic chickens (n  =  50) on Guam and Rota was also assessed. For rails, diagnostic tests included a complete blood count, plasma biochemical analysis, plasma protein electrophoresis, genus-specific plasma enzyme-linked immunoassay (ELISA) for Mycobacterium avium complex (MAC) antigen, arboviral serologic testing, culture for enteric pathogens (Salmonella and Campylobacter species) and Mycobacterium species, fecal examination for parasites, and genus-specific polymerase chain reaction assay for fecal MAC antigen. For chickens, diagnostic tests included arboviral serologic tests and fecal culture for enteric pathogens and Mycobacterium species. Results of testing in the rails were negative for arboviral seroconversion, enteric pathogens, and fecal parasites. Results of screening tests in prerelease rails for Mycobacterium species revealed 16% positive by fecal culture for Mycobacterium species, 0% positive by genus-specific fecal polymerase chain reaction testing for MAC antigen, and 23% seroprevalence by ELISA for genus-specific MAC antigen. Of the 16 positive mycobacterial cultures, 14 (88%) were identified as MAC and 2 (12%) were identified as non-MAC isolates. In chickens, results showed no arboviral seroconversion, no enteric pathogen shedding, and 11% prevalence of fecal non-MAC bacteria.