Amebiasis, a parasitic infection caused by Entamoeba histolytica, is one of the most common parasitic infections worldwide. Since it is still an important public health problem in developing countries, rapid differential diagnosis of amebiasis is crucial in terms of treatment. The most frequently used method for laboratory diagnosis is direct microscopy, however more reliable and specific methods are needed in order to differentiate the apathogenic Entamoeba dispar under the microscope. This study was conducted to compare the results of different methods namely, direct microscopy, culture, ELISA and PCR for the detection of E.histolytica in stool samples and to evaluate the performances of those methods. A total of 1049 stool samples collected from pediatric and adult patients who were admitted to hospital with diarrhea complaint between January 2011-March 2013, and randomly selected samples from primary school children, were included in the study. Direct microscopic examination was performed by native-lugol, physiological saline, modified formol-ethyl acetate sedimentation and trichrome staining methods. The stool samples were also inoculated into TYI-S-33 media for axenic cultivation of amoeba. The presence of amebic antigens in the samples were screened by a commercial ELISA kit (TechLab, E.histolytica II, USA). For the molecular diagnosis, a multiplex tandem real-time PCR (MT-PCR) kit (AusDiagnostics Pty Ltd, Australia) was used, after the extraction of DNAs with QIAamp DNA Stool Mini Kit (Qiagen, USA). A total of 354 samples which could be evaluated by all of the methods, were included in the study. Of the 354 stool samples, 84 (23.7%) were found E.histolytica/E.dispar positive by direct microscopy, 61 (17.2%) by trichrome staining, 46 (12.9%) by culture, 31 (8.7%) by ELISA and 9 (2.5%) by MT-PCR. Of direct microscopy positive samples 54.7% (46/84) were also positive with trichrome staining, 39.3% (33/84) with culture, 15.5% (13/84) with ELISA and 7.1% (6/84) with MT-PCR methods. On the other hand, of the nine MT-PCR positive samples, six were positive with direct microscopy, four with trichrome staining and culture, and one with ELISA. It was remarkable that only one (0.3%) sample yielded positive results with all of the diagnostic methods used. When MT-PCR was considered as the reference method, the sensitivity and specificity values of direct microscopy, trichrome staining, culture and ELISA methods were estimated as; 66.7% and 77.4%, 44.4% and 83.5%, 44.4% and 87.8%, 11.1% and 91.3%, respectively. In conclusion, if the circumstances allow, the use of all methods in combination and evaluation together with the clinical symptoms seems to be the best approaches for the laboratory diagnosis of patients with amebiasis.