The objective of this study was to evaluate the presence of Staphylococcus aureus in the nasal cavity of children attending a daycare center in the town of Francisco Beltrao—PR, by comparing the frequency between age groups and genders. Antimicrobial susceptibility testing with different drugs was also carried out in the bacterial isolates. A prospective study involving 200 children aged 2 and 4 years, grouped by gender, was carried out. Samples were collected from the nasal vestibules of children, incubated and identified. Antimicrobial susceptibility testing was carried out in 30% of the S. aureus colonies. The results showed that 35% of children (10% female and 25% male) were colonized by S. aureus. The frequency of colonization in both male age groups was similar, with 201.93 ± 13.10 CFU/plate for 2-year-old and 266 ± 12.60 CFU/plate for 4-year-old. However, colonization averages for females were different, with 30.43 ± 1.17 CFU/plate for 2-year-old and 394.38 ± 10.70 CFU/plate for 4-year-old. In the antimicrobial susceptibility testing, some strains were resistant to oxacillin, erythromycin and tetracycline, in addition to showing intermediate susceptibility to cephalothin.