【Objective】 To study the inhibitory effect and mechanism of an antimicrobial peptide (A2M3) derived from alpha-2-macroglobulin identified in the human nasal cavity against Staphylococcus aureus. 【Methods】 The mass spectrometry results of the human nasal liquid were analyzed, on the basis of which bioinformatic tools were used for the screening of potential antimicrobial peptides. The minimum inhibitory concentration (MIC) and time-kill curve of A2M3 against S. aureus were determined by the microdilution method and plate colony counting method. Then, transmission electron microscopy, PI uptake assay, flow cytometry, and determination of nucleic acid protein leakage were employed to study the effects of A2M3 on the membrane integrity and permeability of S. aureus. Finally, the effect of A2M3 on the genomic DNA of S. aureus was investigated by the gel retardation assay and fluorescence spectroscopy. 【Results】 A2M3 showed an MIC of 125.0 μg/mL against S. aureus and killed the bacteria completely within 3 h. A2M3 increased the cell membrane permeability to penetrate into S. aureus cells, leading to leakage of nucleic acids and proteins as well as insertion into DNA base pairs to interfere with the gene function, resulting in the death of the cells. 【Conclusion】 The inhibitory mechanism of A2M3 against S. aureus involves multiple targets. The antimicrobial peptide alters the permeability of the bacterial cell membrane and affects the gene function, thus exerting the inhibitory activity. The findings reveal that antimicrobial peptides could be screened and isolated from human body fluids for potential application.