The aim of this study was to compare the mucosal and luminal content microbiota of the cecum and colon of healthy and diarrheic horses. Marked differences in the richness and in the community composition between the mucosal and luminal microbiota of the cecum and large colon of horses with colitis were observed. Microbial dysbiosis occurs in horses with colitis at different levels of the intestinal tract, and microbiota composition is different between the mucosa and luminal content of diarrheic horses. The changes in some key taxa associated with dysbiosis in the equine intestinal microbiota, such as Escherichia, Fusobacterium and Lactobacillus, deserve further inquiry in order to determine their utility for disease diagnosis and treatment.