A new real-time polymerase chain reaction (PCR) method was developed for quantitation of Enterocytozoon bieneusi DNA in sequential stool specimens from immunocompromised patients with intestinal microsporidiosis. Patients were treated with fumagillin (n=6) or with placebo (n=6), in a randomized comparative trial. At baseline, mean E. bieneusi DNA levels were not significantly different in stool specimens from the placebo group, compared with those from the fumagillin group (5.9+/-0.4 vs. 5.9+/-0.6 log(10) copies/microL of stool suspension, respectively; P=.96). In the placebo group, parasitic burden remained stable during follow-up (P=.46), whereas, in the fumagillin group, E. bieneusi DNA levels dropped below the lower limit of detection in all patients (mean reduction from baseline, -4.7 log(10) copies; P<.0001). Real-time PCR performed better than did semiquantitative assessments by microscopy, to measure parasitic burden. In conclusion, this real-time PCR assay is a reliable tool for quantitation of E. bieneusi DNA in stool specimens and for the monitoring of treatment efficacy.