The purpose of this work was to investigate antimicrobial mechanisms of a unique peptide F1 (found in Tibetan Kefir) against Staphylococcus aureus. The cell membrane permeability assay and potassium ion release assay were employed to examine the integrity of the bacterial membrane after the treatment with antimicrobial peptide F1. The transmission electron microscopy was used to analyze the effect of antimicrobial peptide F1 on the cell morphology of Staphylococcus aureus. The interaction between antimicrobial peptide F1 and genomic DNA of Staphylococcus aureus was investigated by DNA gel retardation assay and atomic force microscopy imaging analysis. The results showed that treatment with the antimicrobial peptide F1 led to the permeabilization of the cell membrane of Staphylococcus aureus. Moreover, the antimicrobial peptide F1 can bind to the DNA and inhibit related cellular functions. Having multiple targets means that antimicrobial peptide F1 may be more effective in killing bacterial cells, and bacteria have additional hurdles to overcome to build resistance. These results indicate that antimicrobial peptide F1 is a potential candidate as an effective biological preservative in the agricultural and food industry.