Background <p>Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) is a significant public health concern due to its resistance to multiple antibiotics, primarily mediated by the gene <i>mecA</i>, which encodes penicillin-binding protein 2a (PBP2a), considering the growing global demand for novel antimicrobial strategies.</p> Materials and methods <p>A clinical MRSA strain harboring the gene <i>mecA</i> was identified. Antimicrobial susceptibility testing was performed to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Shirazi thyme (<i>Zataria multiflora</i>) essential oil. The strain’s susceptibility to multiple antibiotics was assessed and the synergistic effect of <i>Z. multiflora</i> essential oil and cefoxitin was also evaluated. Gas chromatography-mass spectrometry (GC-MS) was employed to identify the bioactive compounds in the essential oil. Molecular docking studies were performed to evaluate the competitive binding affinity of those compounds to PBP2x.</p> Results <p>The MRSA strain exhibited resistance to all tested antibiotics except linezolid. The MIC and MBC values for <i>Z. multiflora</i> essential oil were 3.125 mg.mL^− 1 and 6.25 mg.mL^− 1, respectively, for the reference <i>S. aureus</i> strain (ATCC 25923), and 6.25 mg.mL^− 1 for both MIC and MBC for the clinical MRSA strain. The synergy assay demonstrated an enhanced inhibition zone for cefoxitin in combination with <i>Z. multiflora</i> essential oil, indicating a synergistic interaction. Molecular docking studies revealed strong binding interactions between spathulenol, isospathulenol, and aromadendrene comparable to clinically used β-lactam antibiotics.</p> Conclusion <p>The findings highlight the significant antibacterial activity of <i>Z. multiflora</i> essential oil against MRSA and its potential to enhance the efficacy of cefoxitin through synergistic interactions among the inhibitors of natural origin for PBP2x.</p>