A 21-year-old male southern rockhopper penguin (Eudyptes chrysocome) was presented with a chronic history of intermittently decreased appetite, lethargy, and regurgitation. On the external physical examination, the bird was determined to be in fair-to-thin body condition with the complete blood count and plasma chemistry panel being largely unremarkable. Full-body radiographic images were considered normal, and gastroscopy showed only mild gastritis and duodenitis. The penguin was euthanatized shortly thereafter due to acute onset of respiratory distress. During the gross necropsy examination, the bird had severe airsacculitis with thick, yellow-to-tan, moist granular plaques adhering to the surface of many air sacs, as well as regional contiguous pneumonia. Intralesional acid-fast bacilli were observed in histologic sections of air sac tissue, and polymerase chain reaction of the affected air sacs was positive for Mycobacterium fortuitum. This clinical case study describes mycobacteriosis in a sub-Antarctic penguin and to the best of the authors' knowledge, the first reported isolation of M fortuitum from a penguin.