Intestinal amebiasis and gastrointestinal tuberculosis can mimic inflammatory bowel disease and its exacerbations clinically, pathologically, radiologically and endoscopically. In the existence of IBD and/or either one of these two pathogens, early identification and prompt treatment can improve the clinical course of the patients. The aim of this study was to investigate the presence of Entamoeba histolytica and/or Mycobacterium spp. in the first diagnostic biopsy specimens of prediagnosed IBD patients in a tertiary education hospital in Ankara, Turkey. As the differentiation of pathologic Entamoeba histolytica must be based on isoenzymatic, immunologic or molecular analysis and PCR is a rapid and reliable method for the identification of Mycobacterium spp., we investigated the presence of these pathogens in the biopsy specimens of 20 patients who were suspected to have IBD and nine controls, by using PCR-based detection methods. All of them were histopathologically diagnosed as Crohn's disease and none of the specimens contained these two pathogens. We thought that the low prevalence of both infections in Crohn's disease patients may have caused our negative findings and loss of pathogens could have lowered the sensitivity. Further studies with larger number of patients are needed to determine the misdiagnosis rate and coexistence of these three diseases.