A cross-sectional study of Enterocytozoon bieneusi infection in children who lived in an orphanage in Bangkok, Thailand was conducted in April 2003. Two hundred ninety stool specimens were collected and examined under light microscopy after staining with gram-chromotrope. Confirmation of E. bieneusi was done using transmission electron microscopy. Of 290 samples, 12 (4.1%) were positive for E. bieneusi. Genotypic characterization of 10 E. bieneusi showed that all were genotype A, which might indicate the same source of infection. Multivariate analysis showed that orphans who were 12-23 months old, girls, and living in one particular house were independently associated with E. bieneusi infection. Our study suggests that E. bieneusi infection in this orphanage might be transmitted person to person.