A 2-year-old male Humboldt penguin (Spheniscus humboldti) died after a very brief period of illness at a zoo aquarium; the penguin showed sudden depression, anorexia, dyspnoea, and had recurrent melena a day prior to death. The gross examination revealed an extensive bilious effusion in the abdominal cavity due to a gallbladder rupture. Moreover, abscess formation, purulent exudate, severe congestion, and haemorrhages were observed in the trachea and parenchymal organs such as the kidneys and the lungs. A histopathological examination revealed a fibrin deposition with a severe haemorrhage and secondary infiltration of chronic-active inflammatory cells in the parabronchi, atria, and air capillaries and blood vessels of the lungs as well as in most of the parenchymal organs. Moreover, Gram-negative bacilli were found in the lumen of the gastrointestinal tracts including the small and large intestines accompanied by severe epithelial necrosis and the capsule of the liver. Especially, bile pigments were microscopically observed in the whole liver, which indicated a gallbladder rupture. Samples collected from the trachea, lungs, and blood were cultured on a blood agar, and the pure colonies of Proteus genus were isolated. Proteus mirabilis, P. penneri, P. vulgaris, and P. cibarius were identified with polymerase chain reaction (PCR). As a result, the diagnosis was confirmed as Proteus septicaemia. To our knowledge, this is the first report of concomitant infection by different Proteus species that eventually resulted in septicaemia in a Humboldt penguin, and it will provide valuable information for zoo veterinarians for its diagnosis as well, since Humboldt penguins are the most widely found penguins in zoos and Proteus septicaemia in the penguins has, to the best of our knowledge, not been reported as yet.