There is a need to develop feeding strategies to prevent the adverse effect of concentrate feeding in high-performance horses fed energy-dense diets aiming to maintain their health and welfare. The objective of this study is to determine the effect of a VistaEQ product containing 4% live yeast Saccharomyces cerevisiae (S. cerevisiae), with activity 5 × 108 colony-forming unit/g and fed 2 g/pony per day, on faecal microbial populations when supplemented with high-starch and high-fibre diets using Illumina next generation sequencing of the V3-V4 region of the 16S ribosomal RNA gene. The four treatments were allocated to eight mature Welsh section A pony geldings enrolled in a 4-period × 8 animal crossover design. Each 19-day experimental period consisted of an 18-day adaptation phase and a single collection day, followed by a 7-day wash out period. After DNA extraction from faeces and library preparation, α-diversity and linear discriminant analysis effect size were performed using 16S metagenomics pipeline in Quantitative Insights Into Microbial Ecology (QIIME™) and Galaxy/Hutlab. Differences between the groups were considered significant when linear discriminant analysis score was >2 corresponding to P < 0.05. The present study showed that S. cerevisiae used was able to induce positive changes in the equine microbiota when supplemented to a high-fibre diet: it increased relative abundance (RA) of Lachnospiraceae and Dehalobacteriaceae family members associated with a healthy core microbiome. Yeast supplementation also increased the RA of fibrolytic bacteria (Ruminococcus) when fed with a high-fibre diet and reduced the RA of lactate producing bacteria (Streptococcus) when a high-starch diet was fed. In addition, yeast increased the RA of acetic, succinic acid producing bacterial family (Succinivibrionaceae) and butyrate producing bacterial genus (Roseburia) when fed with high-starch and high-fibre diets, respectively. VistaEQ supplementation to equine diets can be potentially used to prevent acidosis and increase fibre digestibility. It may help to meet the energy requirements of performance horses while maintaining gut health.