Shikimic acid (SA) has been reported to possess antibacterial activity against Staphylococcus aureus, whereas the mode of action of SA is still elusive. In this study, the antibacterial activity and mechanism of SA toward S. aureus by cell membrane damage was investigated. After SA treatment, massive K+ and nucleotide leakage from S. aureus, and a significant change in the membrane potential was observed, suggesting SA may act on the membrane by destroying the cell membrane permeability. Through transmission electron microscopic observations we further confirmed that SA can disrupt the cell membrane and membrane integrity. Meanwhile, SA was found to be capable of reducing the membrane fluidity of the S. aureus cell. Moreover, the fluorescence experiments indicated that SA could quench fluorescence of Phe residues of the membrane proteins, thus demonstrating that SA can bind to S. aureus membrane proteins. Therefore, these results showed the antibacterial activity of SA against S. aureus could be caused by the interactions of SA with S. aureus membrane proteins and lipids, resulting in causing cell membrane dysfunction and bacterial damage or even death. This study reveals the potential use of SA as an antibacterial agent.