Citral, a natural antimicrobial agent, has been found to induce severe membrane damage resulting in bacterial death. Despite its effectiveness, there is a lack of in-depth research on its interaction with bacterial cells, especially at low concentrations. This work aimed to investigate the impact of citral concentration on its antibacterial effect against Staphylococcus aureus (S. aureus) and to analyze the relevance between membrane damage and cell death. In this study, the minimal inhibitory concentration of citral against S. aureus was measured at 0.25 mg/mL. At a concentration of 0.5 mg/mL, citral significantly disturbed the cell membrane fluidity and permeability, leading to the leakage of intracellular macromolecules and rapid cell death. However, when the concentration was decreased to 0.25 mg/mL, the membrane damage was greatly reduced, and a complex biological process between citral and membrane components was found. Under continuous drug exposure, the cell membrane underwent gradual rigidification, concomitant with the loss of membrane function, ultimately causing cell death. Despite the fact that no notable disturbance to membrane functions was discovered, the presence of 0.125 mg/mL citral could still weaken the metabolic vitality and growth of S. aureus. Therefore, it was suggested that there could be multiple mechanisms of citral action on S. aureus cells, with drug concentration being a critical factor affecting the mode of action. Overall, the findings of this study provide valuable insights into the antibacterial mechanisms of natural compounds and broaden the potential applications of citral as a natural antimicrobial.