ABSTRACT Investigations regarding Staphylococcus aureus carriage among Brazilian children are scarce. We evaluated the determinants of S. aureus and methicillin-resistant S. aureus (MRSA) nasal carriage in infants attending day care centers (DCCs) and the molecular features of the MRSA strains. A total of 1,192 children aged 2 months to 5 years attending 62 DCCs were screened for S. aureus and MRSA nasal carriage. MRSA isolates were characterized by pulsed-field gel electrophoresis, multilocus sequence typing, spa typing, staphylococcal cassette chromosome (SCC) mec typing and the presence of the Panton-Valentine leukocidin gene. Logistic regression was performed to determine risk factors associated with S. aureus and MRSA colonization. S. aureus and MRSA carriage were detected in 371 (31.1%) and 14 (1.2%) children, respectively. Variables found to be independently associated with an increased risk for S. aureus carriage included being older than 24 months (odds ratio [OR], 1.8; 95% confidence interval [CI], 1.3 to 2.6) and previous DCC attendance (OR, 1.5; 95% CI, 1.0 to 2.2). Having a mother with a high level of education was a protective factor for nasal colonization (OR, 0.4; 95% CI, 0.2 to 0.8). Moreover, we observed that more children carrying MRSA had younger siblings than children not colonized by MRSA. Among the 14 MRSA strains, three SCC mec types (IIIA, IV, and V) were detected, together with a multidrug-resistant dominant MRSA lineage sharing 82.7% genetic similarity with the Brazilian clone (ST239-MRSA-IIIA; ST indicates the sequence type determined by multilocus sequence typing). Although SCC mec type V was recovered from one healthy child who had been exposed to known risk factors for hospital-associated MRSA, its genetic background was compatible with community-related MRSA. Our data suggest that DCC attendees could be contributing to MRSA cross-transmission between health care and community settings.