The objective of this study was to investigate the impact of natural prebiotic active compounds on the microbial composition in different regions of the equine gastrointestinal tract. Twelve adult horses (body weight [bwt] 534 ± 64.5 kg; age 14 ± 7.5 years) were randomly divided into two feeding groups. Six horses received a basal diet consisting of 1.5 kg hay/ 100 kg bwt x d-1 and oat grains equal to 1.19 g starch/kg bwt x d-1, supplemented with Jerusalem artichoke meal providing prebiotic fructooligosaccharides + inulin in a quantity of 0.15 g/kg bwt x d-1. The remaining horses received a placebo added to the basal diet. The horses were fed for 21 d and euthanized at the end of the feeding period. Digesta samples from different parts of the gastrointestinal tract were taken, DNA extracted and the V1-V2 region of the 16S rRNA gene amplified. Supplementation with the prebiotic increased the relative abundance of Lactobacillus (P < 0.05), with a concurrent reduction of the relative abundance of Streptococcus mainly in the stomach (P < 0.05). In the hindgut, the supplemental prebiotic also increased the relative abundance of Lactobacillus but further reduced the relative abundance of fibrolytic bacteria, specifically the unclassified members of the families Lachnospiraceae (P < 0.05) and Ruminococcaceae. The relative abundance of the genus Ruminococcus increased solely in the caecum and colon transversum. Overall, the addition of the prebiotic significantly increased the diversity in nearly all parts of the gastrointestinal tract (P < 0.05). The feeding of this natural prebiotic compound to horses had an impact on the microbial community in the entire gastrointestinal tract. Furthermore, the effect on the bacterial community in the foregut (especially the stomach) was more pronounced in comparison to the effect in the hindgut. Therefore, the impact on stomach health should be carefully considered.