Both oral cavity and subgingival pocket are ecological niches conducive to hosting microorganisms that may act as opportunistic pathogens, such as Staphylococcus aureus and especially methicillin-resistant Staphylococcus aureus (MRSA). Early detection of MRSA is a matter of concern to Public Health. The aim of our study was to determine phenotypic and genotypic detection of methicillin resistance of S. aureus in oral mucosa and subgingival pocket in 102 patients with gingivitis-periodontitis. The prevalence of S. aureus was 10.8% (n = 11) in subgingival pocket and 19.6% (n = 20) in oral mucosa. We obtained 31 isolates of S. aureus of which 13 were mecA positive and 18 were mecA negative. Detection of mecA gene by PCR was used as the reference method to compare the results of phenotypic methods to determine methicillin resistance. Early, accurate detection of S. aureus through phenotyping and genotyping methods is crucial for assessing the colonization and preventing the spread of MRSA.