There is a need to further our understanding of the role that the equine hindgut ecosystem plays in digestive processes and diseases. The aim of the present study was to utilise the real-time PCR technique to determine the abundance of candidate cellulolytic (Ruminococcus flavefaciens; Fibrobacter succinogenes) and non-cellulolytic (Streptococcus bovis) bacteria in lumen contents from the caecum, ventral and dorsal colon, and rectum of healthy horses (n 14). Total DNA was extracted from frozen and lyophilised lumen contents, and PCR primers and Taqman® probes were designed based on 16S rDNA sequences for specific detection of candidate bacterial species. Overall, in frozen and lyophilised digesta, there were significantly (P < 0·01) fewer candidate bacteria in the caecum than the dorsal colon and rectum. In frozen digesta, candidate bacteria levels were similar between the ventral colon, dorsal colon and rectum, but in lyophilised digesta there were significantly (P < 0·05) higher levels of bacteria in the dorsal colon and rectum. Frozen digesta contained disparate levels of candidate bacteria such that R. flavefaciens > F. succinogenes > S. bovis (P < 0·05), while in lyophilised digesta R. flavefaciens was present in significantly (P < 0·05) greater amounts than F. succinogenes and S. bovis. R. flavefaciens and F. succinogenes were abundant at significantly (P < 0·05) greater levels in lyophilised digesta v. frozen digesta, with no difference in S. bovis levels. These data indicate that for these bacteria at least, faeces are a suitable model for studying the bacterial ecosystem within the equine colon. The present study also indicates that the preservation method of digesta affects levels of bacteria detected.