The prevalence of Enterocytozoon bieneusi in the general population is unknown. Using genetic tools, we investigated its prevalence and contribution to diarrhea and malnutrition in hospitalized children in Uganda. A cross-sectional, case-control study involving diarrheic children who were matched for age and sex (3:1) with control children. Measurements included anthropometry and clinical assessment. A total of 17.4% of 1,779 children with diarrhea were infected with E. bieneusi compared with 16.8% of 667 control children (CHI2 = 0.137, P = 0.712). Prevalence was highest during the rainy seasons. There was no significant relationship between infection with E. bieneusi and stunting, being underweight, wasting, or acute diarrhea. However, children who were E. bieneusi-positive by a polymerase chain reaction (PCR) had diarrhea for a longer period (15.15 versus 9.67 days; F = 12.02; P = 0.001) compared with children who were either uninfected or were E. bieneusi-positive by a nested PCR. We conclude that E. bieneusi is widespread among children 3-36 months of age in Uganda, and that in a cross-sectional study, there was no clear association of E. bieneusi with poor nutrition or diarrhea. Since E. bieneusi is closely linked with persistent diarrhea and wasting in adults who are positive for human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), the outcome of follow-up studies involving children who are HIV/AIDS-positive and severely malnourished children may be entirely different and warrants further study.