The short-beaked echidna is the most widespread native Australian mammal and is of ecological and evolutionary importance. Echidnas are also commonly kept in zoological gardens but are often difficult to breed in captivity. The reproductive biology of short-beaked echidnas is unique as one of only three egg-laying mammals (alongside the platypus and long-beaked echidna), where the young hatch from their eggs in the pseudo-pouch in which they reside and are nursed by milk secreted from the milk patch. Previous studies on marsupial pouches have revealed changes in the microbial environment during lactation. To investigate whether the microbiome changes during lactation in echidnas, swab samples were obtained from wild and captive echidna pouches from three different time periods within the reproductive process: outside of breeding season, during courtship and breeding, and during lactation. We found that the pouch microbiome shows dramatic changes during lactation compared to inside and outside of breeding season, with a reduction in bacterial species that may be pathogenic. This suggests that, like marsupials, the pouch environment changes during lactation to accommodate young with an underdeveloped immune system. This study pioneers pouch microbiome research in monotremes, provides new biological information on echidna reproduction, and may inform captive breeding in the future.