Background Horses that undergo surgery for treatment of primary large colon disease have been reported to be at increased risk of developing recurrent colic episodes postoperatively. The reasons for this are currently unknown. The aim of the current study was to characterise the faecal microbiota of horses with colic signs associated with primary large colon lesions treated surgically and to compare the composition of their faecal microbiota to that of a control group of horses undergoing emergency orthopaedic treatment. Faecal samples were collected from horses in both groups on admission to hospital, during hospitalisation and following discharge from hospital for a total duration of 12 weeks. Additionally, colonic content samples were collected from surgical colic patients if pelvic flexure enterotomy was performed during laparotomy. A total of 12 samples were collected per horse. DNA was extracted from samples using a commercial kit. Amplicon mixtures were created by PCR amplification of the V1 – V2 regions of the bacterial 16S rRNA genes and submitted for sequencing using the Ion Torrent PGM next-generation sequencing system. Multivariate data analysis was used to characterise the faecal microbiota and to investigate differences between groups. Results Reduced species richness was evident in the colonic samples of the colic group compared to concurrent sampling of the faeces. Alpha and beta diversity differed significantly between the faecal and colonic microbiota with 304 significantly differentially abundant OTUs identified. Only 46 OTUs varied significantly between the colic and control group. There were no significant differences in alpha and beta diversity of faecal microbiota between colic and control horses at admission. However, this lack of significant differences between groups should be interpreted with caution due to a small sample size. Conclusions The results of the current study suggest that faecal samples collected at hospital admission in colic cases may not accurately represent changes in upper gut microbiota in horses with colic due to large colon disease.