Dientamoeba fragilis is a protozoan parasite of humans that infects the mucosa of the large intestine and is associated with gastrointestinal disease. We developed a 5' nuclease (TaqMan)-based real-time PCR assay, targeting the small subunit rRNA gene, for the detection of D. fragilis in human stool specimens and compared its sensitivity and specificity to conventional PCR and microscopic examination by a traditional modified iron-hematoxylin staining procedure. Real-time PCR exhibited 100% sensitivity and specificity.