Infectious agents have the potential to thrive in child daycare facilities. Asymptomatic Staphylococcus aureus carriage is a risk factor for developing infection and contributes to transmission. We collected swabs from 110 employees, 111 unexposed adults, 81 children, and 214 environmental surfaces at 11 Iowa daycare facilities. S aureus isolates were characterized using antibiotic resistance profiles and Staphylococcal protein A typing. Staphylococcal protein A types were grouped into cluster complexes using the Based Upon Repeat Pattern algorithm. All isolates (from 38 employees, 37 unexposed adults, 16 children, and 19 surfaces) were characterized. Daycare employees were more likely to carry erythromycin-resistant S aureus than unexposed adults (odds ratio, 3.7; 95% confidence interval, 1.1-12.7; P=.033). Isolates were genetically heterogeneous, although isolates from employees appeared more clonal than those from unexposed adults. Strains associated with ST8 were identified in 5 daycare facilities and 3 unexposed adults. S aureus isolates collected from employees, children, and surfaces of daycare facilities are genetically heterogeneous, but contain strains associated with community-associated methicillin-resistant S aureus. This suggests that daycare facilities can serve as reservoirs for community-associated methicillin-resistant S aureus and facilitate genetic exchange. Employees may be at increased risk of carrying antibiotic-resistant strains, indicating more research is necessary into this occupational group.