Mycobacterium intracellulare (MIT) was diagnosed postmortem by culture and supporting histopathology in seven birds from a flock of little blue penguins (Eudyptula minor) at the Henry Doorly Zoo (HDZ). These birds represented 20% of the deaths in the population over a 4 yr period. Clinical signs in affected birds included severe respiratory distress characterized by open-mouth breathing with chronic debilitation. On exam, plaques were noted in the larynx, trachea, and soft tissue of the caudal oropharynx. Index cases were identified on necropsy in two birds on loan to another institution in 2003. Following a case confirmed antemortem at the HDZ, a three-drug protocol of rifampin (15 mg/kg p.o. s.i.d.), ethambutol (15 mg/kg p.o. s.i.d.), and clarithromycin (10 mg/kg p.o. s.i.d.) was started on this bird in 2004 and extended to the entire flock in 2005. Gastric wash, fecal samples, and throat plaques were obtained antemortem on five birds within the flock, selected because of the presence of oral plaques, and tested by culture followed by a polymerase chain reaction assay. MIT was detected in gastric washes from four birds and in throat plaques from all five. Three more birds died during treatment. After the seventh bird died, antimicrobial susceptibility testing performed in July 2007 indicated that the MIT was now resistant to most antibiotics tested, including rifampin and ethambutol. The treatment regimen was changed to minocycline (10 mg/kg p.o. b.i.d.) and clarithromycin (10 mg/kg p.o. s.i.d.). Oral plaques were not seen on monthly rechecks of the flock through November 2008. The proposed mechanism of transmission is exposure to wild birds but the source has not been determined. These cases of avian mycobacteriosis caused by MIT are the first known cases reported in little blue penguins.