The mule deer in arid regions of northern Mexico hold ecological and hunting significance. Their gastrointestinal microbiota plays a key role in health and energy extraction. This study characterises and compares the faecal bacterial profile and function of semi-captive (DS) and wild (DW) mule deer (Odocoileus hemionus crooki) populations. This was achieved by sequencing the V3–V4 region of the 16S rRNA gene. Functional bacterial predictions were evaluated based on Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) analysis. The phyla Firmicutes (DS, 63.8%; DW, 40.4%), Bacteroidetes (DS, 25.4%; DW, 35.5%), and Proteobacteria (DS, 8.8%; DW, 10.7%) were more abundant in all samples, where Firmicutes was more abundant in the DS and Bacteroidetes in DW. Differences were observed in beta diversity between populations, with Firmicutes, Planctomycetes, Christensenellaceae and other taxa, contributed to the dissimilarity. A total of 352 metabolic pathways were identified, where cellular metabolism, unsaturated fatty acid metabolism, and amino acid metabolism were the enriched pathways. There were no significant differences in functional pathways between DS and DW populations. Bacterial communities in mule deer are mainly diet-associated, contributing to wildlife microbiome research. These insights have the potential to improve conservation efforts and promote deeper research into the cervid microbiota worldwide.