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Background In the past decade methicillin-resistant Staphylococcus aureus (MRSA) has become increasingly prevalent in community settings. Attending a daycare center (DCC) is a known risk factor for colonization with MRSA. Brazil operates free, public DCCs for low-income families, some of which are located in census tracts defined by the Brazilian Census Bureau as informal settlements (aglomerados subnormais, AGSN). Physical and demographic characteristics of AGSNs suggest that S. aureus colonization prevalence would be higher, but little is known about the prevalence of MRSA in these settings. Methods We conducted a cross-sectional study to assess risk factors for S. aureus and MRSA colonization among children attending DCCs located in AGSN vs non-AGSN. Nasal swabs were collected from children aged three months to six years in 23 public DCCs in Niterói, Brazil between August 2011 and October 2012. Results Of 500 children enrolled in the study, 240 (48%) were colonized with S. aureus and 31 (6.2%) were colonized with MRSA. Children attending DCCs in AGSNs were 2.32 times more likely to be colonized with S. aureus (95% CI: 1.32, 4.08), and 3.27 times more likely to be colonized with MRSA than children attending non-AGSN DCCs (95% CI: 1.52, 7.01), adjusted for confounding variables. Conclusion S. aureus and MRSA colonization prevalence among children attending DCCs in informal settlement census tracts was higher than previously reported in healthy pre-school children in Latin America. Our data suggest that transmission may occur more frequently in DCCs rather than at home, highlighting the importance of DCCs in AGSNs as potential MRSA reservoirs. This finding underscores the importance of local epidemiologic surveillance in vulnerable AGSN communities. Electronic supplementary material The online version of this article (doi:10.1186/1471-2334-14-538) contains supplementary material, which is available to authorized users.