Staphylococcus aureus is a prevalent foodborne pathogen. However, it has exhibited increasing tolerance to common preservatives. The aim of this study was to analyze new natural preservatives to combat this issue. In this study, sanggenon C and D, derived from mulberry trees, were investigated to understand the inhibition mechanisms involved in the cell membrane biosynthesis of S. aureus by using scanning electron microscopy, quantitative real-time PCR, and parallel reaction monitoring. The results indicated that the minimum inhibitory concentrations of sanggenon C and sanggenon D against S. aureus were 70.60 μmol•L-1 and 17.60 μmol•L-1, respectively, demonstrating significantly superior efficacy when compared with that of cinnamaldehyde. Furthermore, sanggenon D disrupted the cell structure and morphology of S. aureus. Notably, sanggenon D exhibited a stronger inhibitory effect on S. aureus than sanggenon C for the expression of genes and proteins related to phospholipid biosynthesis. In particular, sanggenon D exerted a more significant inhibitory effect on the expressions of Fab H and CdsA within the phospholipid synthesis pathway. Based on molecular docking results, the interaction between sanggenon D and PlsX probably involved the formation of hydrogen bonds at Lys290, Glu233, Gly293, and Ser294, with a greater interaction strength than that of the hydrogen bond interaction between sanggenon C and PlsX. This mechanism may account for the enhanced inhibitory effect of sanggenon D when compared with that of sanggenon C. Therefore, sanggenon D holds potential for development as a natural food preservative. These findings offer a theoretical foundation for the application of sanggenon D as a novel type of natural preservative.