Background & objective: Photodynamic inactivation (PDI) has been investigated to cope with the increasing incidence of multidrug-resistant (MDR) pathogens. PDI employs nontoxic photosensitizers (PSs), which localize in the microbial cells and are activated by a specific wavelength of visible light. The main purpose of this study was to explore the PDI effect of methylene blue (MB) and toluidine blue O (TBO) on Escherichia coli (ATCC 25922) and clinical isolate of multidrug-resistant Escherichia coli.Methods: Effect of PSs concentration (12.5, 25, 50 µg/ml) and laser irradiation time (10, 20, 30 min) on lethal photosensitization was investigated.Results: Methylene blue (50 µg/ml) photosensitization using red laser light (163.8 J/cm2) was able to achieve reductions of 53.1% and 37.6% in the viable counts of Escherichia coli (ATCC 25922) and clinical isolate of MDR Escherichia coli (using starting concentrations of 104–105 CFU ml-1). TBO at 50 µg/ml, 46.8 Jcm-2, exhibited 0.7 log killing for MDR E. coli and 1.7 log killing for E. coli (ATCC 25922).Conclusion: In our study, the selected MDR isolate was more resistant to TBO-PDI-mediated killing than its ATCC reference strain. Therefore, the PDI efficacy of TBO may be affected by the antibiotic resistance mechanism that presented in MDR isolate.