Abstract Koalas ( Phascolarctos cinereus ) are iconic Australian marsupials currently threatened by several processes. Infectious reproductive tract disease, caused by Chlamydia pecorum , and koala retrovirus infection are considered key drivers of population decline. The clinical sign of ‘wet bottom’, a staining of the rump associated with urinary incontinence, is often caused by chlamydial urogenital tract infections. However, wet bottom has been recorded in koalas free of C. pecorum , suggesting other causative agents in those individuals. Current understanding of the bacterial community of the koala urogenital tract is limited. We used 16S rRNA diversity profiling to investigate the microbiome of the urogenital tract of ten female koalas. This was to produce baseline data on the female koala urogenital tract microbiome, and to undertake preliminary investigations of potential causative agents of wet bottom, other than C. pecorum . Five urogenital samples were processed from koalas presenting with wet bottom and five were clinically normal. We detected thirteen phyla across the ten samples, with Firmicutes occurring at the highest relative abundance (77.6%). The order Lactobacillales , within the Firmicutes , comprised 70.3% of the reads from all samples. After normalising reads using DESeq2 and testing for significant differences ( P < 0.05), there were 25 operational taxonomic units (OTUs) more commonly found in one group over the other. The families Aerococcaceae and Tissierellaceae both had four significantly differentially abundant OTUs. These four Tissierellaceae OTUs were all significantly more abundant in koalas with wet bottom. Importance This study provides an essential foundation for future investigations of both the normal microflora of the koala urogenital tract, and better understanding of the causes of koala urogenital tract disease. Koalas in the states of Queensland and New South Wales are currently undergoing decline, and have been classified as vulnerable populations. Urogenital tract disease is a leading cause of hospital admissions in these states, yet previously little was known of the normal flora of this site. Wet bottom is a clinical sign of urogenital tract disease, which is often assumed to be caused by C. pecorum and treated accordingly. Our research highlights that other organisms may be causing wet bottom, and these potential aetiological agents need to be further investigated to fully address the problems this species faces.