Background Clinical significance of Staphylococcus aureus colonization has been demonstrated in hospital settings; however, studies in the community have shown contrasting results regarding the relevance of colonization in infection by community-associated MRSA (CA-MRSA). In Colombia there are few studies on S. aureus colonization. The aim of this study was to determine the molecular and epidemiological characteristics of nasal colonization by S. aureus (MSSA-MRSA) in children from a university hospital and day care centers (DCCs) of Medellin, Colombia. Methods An observational cross-sectional study was conducted in 400 children (200 in each setting), aged 0 months to 5 years, during 2011. Samples were collected from each nostril and epidemiological information was obtained from the parents. Genotypic analysis included spa typing, PFGE, MLST, SCCmec typing, detection of genes for virulence factors and agr groups. Results Frequency of S. aureus colonization was 39.8% (n = 159) (hospital 44.5% and DCCs 35.0%) and by MRSA, 5.3% (n = 21) (hospital 7.0% and DCCs 3.5%). Most S. aureus colonized children were older than two years (p = 0.005), the majority of them boys (59.1%), shared a bedroom with a large number of people (p = 0.028), with history of β-Lactamase inhibitors usage (p = 0.020). MSSA strains presented the greatest genotypic diversity with 15 clonal complexes (CC). MRSA isolates presented 6 CC, most of them (47.6%) belonged to CC8-SCCmec IVc and were genetically related to previously reported infectious MRSA strains. Conclusion Differences in epidemiological and molecular characteristics between populations may be useful for the understanding of S. aureus nasal colonization dynamics and for the design of strategies to prevent S. aureus infection and dissemination. The finding of colonizing MRSA with similar molecular characteristics of those causing infection demonstrates the dissemination capacity of S. aureus and the risk of infection among the child population.