BACKGROUND Bacterial resistance to antibiotics is generally increasing, which has become a great challenge for treating infectious diseases caused by microbes. Photodynamic antibacterial chemotherapy (PACT) has been considered as a promising method for inactivating bacteria. The combination of antimicrobial agent with PACT may provide efficient way against drug-resistant microbe. This study aims to investigate the synergistic effects of PACT mediated by toluidine blue (TB), combined with gentamicin (GEN) on common pathogens Staphylococcus aureus (S. aureus) and multidrug-resistant S. aureus (MDR S. aureus). METHODS Alkaline lysis was used to detect the uptake of TB by S. aureus and MDR S. aureus. Plate counting was applied to evaluate the inhibition efficiency of GEN alone, TB-PACT alone, and work together. Flow cytometry and fluorescence microscopy were performed to examine the permeability of bacterial membranes after different treatments. Intracellular and extracellular reactive oxygen species (ROS) were assessed with the assist of H2DCF-DA and SOSG probes. RESULTS TB-PACT combined with GEN led to more pronounced antibacterial effects in S. aureus and MDR S. aureus, as compared with either alone. TB-PACT treatment permeabilized the bacterial membranes, promoted GEN cellular accumulation and augmented the antibacterial efficiency. The intracellular ROS generation by the combination of TB-PACT and GEN was much higher than that of single treatment groups. CONCLUSIONS TB-PACT decreased the GEN cytotoxic threshold and usage, and the synergy of them significantly enhanced the sterilization of S. aureus and MDR S. aureus.