Investigating PFM01's bactericidal mechanism reveals its impact on the permeability and fluidity of S. aureus cell membranes, suggesting targeted effects on specific membrane components. The checkerboard method and subsequent proteomic and whole-genome sequencing analyses under PFM01 pressure elucidate alterations in membrane phospholipids, up-regulation of transmembrane and membrane-associated proteins, and identification of mutations in resistant strains, providing insights into PFM01's antibacterial action.