The granules of circulating leukocytes contain reactive oxygen species that are important components of host defence against bacterial invasion. We report the capacity of marsupials and monotremes to mount such a defence in a manner similar to their eutherian relatives. Using the nitroblue tetrazolium (NBT) test, reactive oxygen species were detected in the peripheral blood cells of five captive marsupial species (the tammar wallaby, Macropus eugenii, the Rufous hare wallaby, Lagorchestes hirsutus, the Brush-tailed bettong, Bettongia penicillata, the Long-footed potoroo, Potorous longipes, and the Long-nosed potoroo, Potorous tridactylus). The study included animals that were clinically healthy and those that were affected by mycobacterial disease. Animals in poor health elicited weak responses, consistent with the NBT test being used as a diagnostic assay for immunodeficiency. The NBT slide assay was also applied to platypus (Ornithorhyncus anatinus) and short-beaked echidna (Tachyglossus aculeatus) granulocytes to confirm the applicability of the test to this unique group of mammals.