Entamoeba histolytica, Giardia lamblia, and Cryptosporidium are three of the most important diarrhea-causing parasitic protozoa. For many years, microscopic examination of stool samples has been considered to be the "gold standard" for diagnosis of E. histolytica, G. lamblia, and C. parvum infections. Recently, more specific and sensitive alternative methods (PCR, enzyme-linked immunosorbent assay, and direct fluorescent-antibody assay) have been introduced for all three of these parasitic infections. However, the incorporation in a routine diagnostic laboratory of these parasite-specific methods for diagnosis of each of the respective infections is time-consuming and increases the costs of a stool examination. Therefore, a multiplex real-time PCR assay was developed for the simultaneous detection of E. histolytica, G. lamblia, and C. parvum in stool samples. The multiplex PCR also included an internal control to determine efficiency of the PCR and detect inhibition in the sample. The assay was performed on species-specific DNA controls and a range of well-defined stool samples, and it achieved 100 percent specificity and sensitivity. The use of this assay in a diagnostic laboratory would provide sensitive and specific diagnosis of the main parasitic diarrheal infections and could improve patient management and infection control.