Asymptomatic carriage of Staphylococcus aureus is an important risk factor for developing infection, as well as a key contributor to transmission. Despite the fact that childcare workers are at known risk for infections, little research has focused on bacterial carriage in this occupational group. We conducted a cross-sectional study comparing the prevalence of S. aureus in 110 childcare employees with the prevalence in 111 unexposed participants. Questionnaire data was collected for employees, unexposed adults, and children. Multivariate analysis allowed for adjustment for confounders. Data on work-related exposures are presented as well. The prevalence of S. aureus in employees, unexposed adults, children, and on surfaces was 35·2%, 33·6%, 19·8%, and 9·8%, respectively. Washing children's hands upon their arrival at childcare facilities may decrease the risk of S. aureus carriage in employees (odds ratio 0·17, 95% confidence interval 0·095–0·32, P < 0·0001).