Background <p>The gut microbiome is an ecosystem of microbes that live within the host’s digestive tract and are a vital component of host health. An increasing number of studies are investigating microbiome differences in wild and captive-managed populations to assist in species conservation and improve captive welfare. The gut microbiota of Australian marsupials has not been extensively studied but has long been recognised as having an important functional role in the digestive physiology and health of those in care. We used 16S rRNA gene sequencing to characterise and compare the faecal bacterial communities of six species of Australian marsupials from captive or wild origins over a large temporal and spatial scale.</p> Results <p>We found the microbiome of captive marsupials had reduced microbial richness and diversity in two species, the brushtail possum and the Eastern grey kangaroo. Captivity was associated with gut microbiome compositional differences for half of the species tested. These compositional changes were accompanied by less pronounced seasonal variability in captivity.</p> Conclusions <p>This study provides valuable baseline data and demonstrates that captivity significantly alters the gut microbiota, suppressing its natural seasonal variability. These findings enhance our understanding of the gut microbiome in Australian marsupials. Future research should focus on determining the functional importance of these microbial communities and develop strategies to address any microbiome deficiencies in managed populations. Such efforts could ultimately improve the success of captive rearing and reintroduction programs.</p>