The intestinal microbiota, an invisible organ supporting a host’s survival, has essential roles in metabolism, immunity, growth, and development. Due to the influence of the intestinal microbiota on the biology of an organism, application of such data to wildlife conservation has gained interest. While there is a standard protocol for studying the human intestinal microbiota, an equivalent one for wildlife is still under development. A major challenge is sampling the intestinal microbiota in an effective, unbiased way. Faecal samples are a popular proxy of the intestinal microbiota because collection is non-invasive, convenient, and allows for longitudinal sampling. Yet, it is unclear whether the faecal microbiota is representative of the intestinal microbiota. In amphibians, research on sampling methodology is limited. In this study, we first characterise and compare microbiota (small intestine, large intestine, faeces) of two Hong Kong stream-dwelling frog species: the Lesser Spiny Frog (Quasipaa exilispinosa) and the Hong Kong Cascade Frog (Amolops hongkongensis). We found that both species have similar dominant phyla and families, but diverge in terms of the dominant genera. Second, we assess the performance of faecal microbiota in representing the intestinal microbiota in these two species. By comparing the microbiota between three sample types (small intestine, large intestine, and faeces), we found (1) that microbiota of small and large intestine differs significantly, (2) that faeces are not an appropriate proxy of both intestinal sections, and (3) a set of microbial taxa that significantly differ between sample type. Our study raises caution on equating faecal and intestinal microbiota. The use of faeces can avoid sacrifice an animal, but researchers should avoid over-extrapolation and interpret results carefully.