Tuberculosis (TB) patients are normally treated with a combination of antibiotics. However, with improper or incomplete treatment of antibiotics, the disease may progress to multidrug-resistant TB (MDR-TB). The treatment of MDR-TB is very costly and inefficient. Therefore, there is a great demand of new therapeutic approaches for MDR-TB such as photodynamic therapy. In this study, we tried to optimize the conditions for photodynamic inactivation of TB using methylene blue as a photosensitizer. Different combinations of methylene blue concentrations and light doses were tested for their photodynamic effects to A549 cells or Mycobacterium smegmatis (M. smegmatis). We also tested the effect of photodynamic therapy on ciprofloxacin-resistant M. smegmatis. Methylene blue treatment alone did not affect the survival rates of A549 cells or bacteria up to 5 μg/ml. When the A549 and M. smegmatis cells treated with methylene blue were irradiated with laser light (wavelength, 630 nm), photodynamic inactivation of cells was increased in methylene blue concentration-and light dose-dependent manners. Interestingly, the ciprofloxacin-resistant M. smegmatis exhibited higher level of susceptibility to methylene blue-mediated photodynamic inactivation. This study suggests that photodynamic therapy at 3.6 J/cm 2 in the presence of 5 μg/ml methylene blue may be an appropriate range for therapy due to the high bactericidal activity against high level of ciprofloxacin-resistant M. smegmatis and the low damaging effect to mammalian cells. This study demonstrates that photodynamic therapy could be a potential alternative for MDR-TB treatment.