Abstract: OBJECTIVE To investigate the bactericidal effects of photodynamic antimicrobial chemotherapy (PACT) on three common multidrug-resistant (MDR) bacteria, including methicillin-resistant Staphylococcus aureus(MRSA), multidrug-resistant Pseudomonsa aeruginosa(MDRP) and ESBLs-producing Escherichia coli(ESBLs⁺ E.coli), and the effects on the formation of biofilms. METHODS The optimal doses of light intensity and toluidine blue O (TBO) were determined using three MDR bacteria. Survival of bacteria and value of killing logarithm(KL) were measured to determine the antimicrobial efficacy of TBO-PACT on these MDR bacteria, and the sensitivity of bacteria to TBO-PACT were also detected. Gram staining and environmental scanning electron microscope (ESEM) were used to determine the antimicrobial efficacy of TBO-PACT for the biofilms of MRSA and MDRP. RESULTS The killing effects of single laser irradiation on 3 drug-resistant strains were weak, and showed a certain enhancement with the increase of laser intensity, and TBO could increase the killing effect of bacteria (P<0.001). However, with the increase of TBO concentration, the killing rates of the 3 drug resistant strains in the experimental group increased first and then decreased, and reached the peak value at TBO concentration of 16.67 μg/ml. The order of sensitivity of 3 drug resistant bacteria to PACT was as the following: MRSA> ESBLs⁺ E.coli> MDRP. The results with light and electron microscope suggested that the formation of MRSA and MRDP bacterial biofilms could significantly be inhibited by PACT. CONCLUSION PACT is a potential treatment method for MDR bacterial infections. PACT would have opportunity to apply to clinical in refractory traumatic infection and can reduce the incidence of bacterial resistance in hospital in the future.