European ungulates such as the roe deer face seasonally varying climatic conditions as well as food availability and quality. In some European countries, including Austria, it is common practice to provide game animals with supplemental feeding in winter. In this study we investigated if supplemental feeding significantly affects the composition of the bacterial rumen community. The rumen microbial composition of eight adult female roe deer was analysed by Illumina MiSeq 16S rRNA gene amplicon sequencing. Animals from a study area with supplemental feeding sites were compared to individuals relying on natural feed. Furthermore, the microbial community composition of different ruminal compartments (liquid phase, solid phase and wall) was compared. Our results revealed a significant qualitative difference between the microbiota composition of the two populations studied. Easily fermentable supplemental feeding promoted the proliferation of phylotypes correlated with conditions of acidosis in domestic ruminants, suggesting a possible similar adaptation and a hypothetical negative effect on health status also in roe deer. The results furthermore confirmed that in roe deer, like in other ruminant species, the most represented phyla are Firmicutes (63.2%) and Bacteroidetes (23.5%), and that the ruminal microenvironments influence the microbial community composition, with the lowest species richness and variation in the epimural microbiota.