A group of zoo-housed little penguins (Eudyptula minor) was diagnosed with mycobacteriosis. While undergoing multidetector computed tomography (MDCT) imaging for an unrelated research project, pulmonary lesions were detected in multiple individuals. In general, birds appeared healthy and free of outward signs of disease. After the loss of three individuals, polyclonal mycobacterial disease due to Mycobacterium avium-intracellulare complex was confirmed. Surviving birds were treated with rifampin (45 mg/kg), ethambutol (30 mg/kg), clarithromycin (10 mg/kg), and enrofloxacin (30 mg/kg) compounded into a single capsule administered once a day in food. After 3 mo of therapy, MDCT imaging documented a decrease in nodule size and number in all remaining birds, with further improvement documented after 13 mo of treatment. MDCT imaging was invaluable for diagnosing disease, documenting disease progression over time, and assessing response to therapy. Early initiation of therapy before the development of outward signs of disease led to resolution of mycobacterial pulmonary lesions in multiple penguins. Mycobacterial disease in this group of little penguins, as well as previously published reports, suggests that the species is at increased risk for developing mycobacteriosis.