Chlamydia trachomatis is frequently detected in anorectal specimens from men and women. A recent hypothesis suggests that C. trachomatis is a natural commensal organism asymptomatically colonizing the gastrointestinal tract. In this study, we investigated the presence of chlamydial DNA and antigen in intestinal biopsy samples taken during colonoscopy. Cases (n = 32) were patients whose histopathology reports included the term 'chlamydia', suggesting a possible history of infection. Control patients (n = 234) did not have chlamydia mentioned in their histopathology report and all tested negative for Chlamydiaceae DNA by 23S ribosomal RNA-based real-time PCR. Amongst the cases, C. trachomatis DNA was detected in the appendix and colon of two female and one male patients. Chlamydia abortus DNA was present in the colon of a fourth female patient. Thus, chlamydial DNA could be demonstrated in intestinal biopsy samples proximal to the anorectal site and inclusions were identified in rectum or appendix of two of these patients by immunohistochemistry. However, the findings in two cases were compatible with sexually acquired C. trachomatis. The identification of C. trachomatis DNA/antigen does not prove the presence of active infection with replicating bacteria. Larger prospective studies on fresh tissue samples are required to confirm the data obtained in this study.