Juglone is a plant-derived 1,4-naphthoquinone with confirmed antibacterial activity. However, the mechanism of action of juglone against Staphylococcus aureus remains unclear. Possible mechanisms were explored by a proteomic analysis of S. aureus proteins that are inhibited by juglone. Two-dimensional gel electrophoresis revealed that 21 protein spots were differentially expressed between juglone-treated and untreated cells of which 13 were identified. A bioinformatic analysis revealed that proteins participating in protein synthesis, the tricarboxylic acid cycle, as well as DNA and RNA synthesis were inhibited by juglone, thus leading to cell collapse. These findings provide clues regarding the mechanism of action of juglone, which can be effective for treating cases of S. aureus infection.