Background: Children attending day care centres are at increased risk of infectious diseases, in particular gastrointestinal and respiratory infections. Hand hygiene of both caregivers and children is an effective prevention measure. This study examined hand hygiene behaviour of children attending day care centres, and describes hygiene facilities at day care centres. Methods: Data were collected at 115 Dutch day care centres, among 2318 children cared for by 231 caregivers (August to October 2010). Children's hand hygiene behaviour was observed and data on hand hygiene facilities of the day care centres collected by direct unobtrusive observation. National guidelines indicate hand hygiene is required before eating, after toilet use and after playing outside. Results: Among 1930 observed hand hygiene opportunities for children, overall adherence to hand hygiene guidelines was 31% (95% CI: 29-33%). Adherence after both toilet use and playing outside was 48%. Hands were less frequently washed before eating, where guideline adherence was 15%. In 38% of the playrooms there was no soap within reach of children and 17% had no towel facilities. In over 40% of the playrooms, appropriate hand hygiene facilities for children were lacking. Conclusion: Adequate hand washing facilities were available for children in only half of the participating day care centres in our study and children washed their hands in only 15-48% of the occasions defined by official guidelines. More attention is needed to hand hygiene of children attending day care centres in the prevention of infectious diseases.