Nasal carriage of Staphylococcus aureus by healthcare workers is of great clinical importance as it facilitates the contamination of medical devices and cross-transmission. However, studies regarding the epidemiology and dissemination of S. aureus and Methicillin-resistant S. aureus (MRSA) within the Primary Health Care in Brazil are scarce. The current study aimed to detect and characterize S. aureus and MRSA strains from the nasal cavities of 63 healthcare working in primary health care units in order to determine the prevalence of S. aureus and MRSA, biofilm formation and resistance profile of these isolates. PCR reactions were performed for detecting mecA, icaA and icaD genes. The phenotypic antimicrobial susceptibility was assessed by the disk diffusion method and biofilm formation by the Congo Red Agar (CRA) method. The MRSA isolates were typed for the Staphylococcal Cassette Chromosome mec (SCCmec). The prevalence of nasal carriage of S. aureus was 74.6%, of which 72.3% were MRSA carrying SCCmec type I (24.4%), III (34.1%), IV (36.6%). Two (4.9%) isolates presented a non-typeable cassette by the performed technique. The antimicrobial susceptibility evaluation evidenced penicillin resistance in 66.1% of S. aureus, erythromycin resistance in 49.2%, while 37.3% were resistant to oxacillin, 28.8% to cefoxitin, 5.1% to levofloxacin and 5.1% to clindamycin. All isolates were biofilm producers and 96.6% of the strains contained the ica biofilm-forming genes (icaA and/or icaD). We have demonstrated a high prevalence of S. aureus and MRSA carriage among health care working in Primary Health Care units, the presence of SCCmec types I, III and IV, in addition to their high ability to form biofilm, factors that possibly contribute to the dissemination and persistence of these pathogens within the primary care services. These observations highlight the importance of broadening the perspective of Health Care-Associated Infections prevention, including all health care levels, which are currently little explored. In addition, the dynamics and resistance mechanisms of S. aureus transmission still need to be further clarified to enable the implementation of more effective prevention measures.