Staphylococcus aureus is one of the most serious pathogens threatening food safety and public health. We have previously showed that iturin W exhibited obvious antifungal activity on plant pathogens. In the present study, we found iturin W, especially C14 iturin W, showed strong antimicrobial activity against S. aureus, and the antimicrobial mechanism of C14 iturin W was further investigated by transcriptomic analysis and a related biochemical experiment. The results showed that C14 iturin W can reduce the expression levels of genes associated with the reactive oxygen species (ROS) scavenging enzyme and genes involved in arginine biosynthesis, thus leading to the increase in ROS levels of S. aureus. Furthermore, C14 iturin W can also interfere with proton dynamics, which is crucial for cells to regulate various biological possesses. Therefore, ROS accumulation and change in proton motive force are import ways for C14 iturin W to exert the antimicrobial activity. In addition, C14 iturin W can also reduce the expression levels of genes related to virulence factors and decrease the production of enterotoxins and hemolysins in S. aureus, indicating that C14 iturin W has a good potential in food and pharmaceutical fields to reduce the harm caused by S. aureus in the future.