Non-invasive sampling methods for studying intestinal microbiome are widely applied in studies of endangered species and in those conducting temporal monitoring during manipulative experiments. Although existing studies show that non-invasive sampling methods among different taxa vary in their accuracy, to date, no studies have been published comparing non-lethal sampling methods in adult amphibians. In this study, we compare microbiomes from two non-invasive sample types (faeces and cloacal swabs) to that of the large intestine in adult cane toads, Rhinella marina. We use 16S rRNA sequencing to investigate how microbial communities change along the digestive tract and which non-lethal sampling method better represents large intestinal microbiota. We found that cane toads' intestinal microbiota was dominated by Bacteroidetes, Proteobacteria, Firmicutes and, interestingly, we also saw a high proportion of Fusobacteria, which has previously been associated with marine species and changes in frog immunity. The large and small intestine of cane toads had a similar microbial composition, but the large intestine showed higher diversity. Our results indicate that cloacal swabs were more similar to large intestine samples than were faecal samples, and small intestine samples were significantly different from both non-lethal sample types. Our study provides valuable information for future investigations of the cane toad gut microbiome and validates the use of cloacal swabs as a non-lethal method to study changes in the large intestine microbiome. These data provide insights for future studies requiring non-lethal sampling of amphibian gut microbiota.