Purpose: We evaluate the antimicrobial effect of toluidine blue O (TBO)-mediated photodynamic antimicrobial chemotherapy (PACT) on Staphylococcus epidermidis and Staphylococcus aureus isolated from ocular surface infection. Methods: We selected 24 strains of bacteria for this study. The antimicrobial effect of different TBO concentrations, light irradiation, and duration were evaluated. After determining the optimal PACT parameters, four experimental groups were included: Group 1, TBO alone (T+L-); Group 2, light-emitting diode (LED) irradiation alone (T - L+); Group 3, TBO-LED irradiation combination (T+L+); and Group 4, no treatment group (T-L- ). The antibacterial effect of PACT was evaluated with the colony survival fraction (SF) methods. Results: The antibacterial effect of PACT on S. epidermidis and S. aureus was dose-dependent to light irradiation and TBO concentration. The optimal PACT parameters were a TBO concentration of 60 mu M, light irradiation of 5.27 mW/cm(2), and an irradiation duration of 30 minutes. In group 1, 60 mu M TBO without irradiation did not show any antibacterial effect on S. epidermidis (1.48E7 +/- 1.5E6 colony-forming units [CFU]/mL) or S. aureus (1.45E7 +/- 9E5 CFU/mL). In group 2, irradiation alone with 5.27 mW/cm(2) did not modify bacterial growth (S. epidermidis, 1.49E7 +/- 1.43E6; S. aureus, 1.5E7 +/- 1.2E6). In group 3, after treatment, bacteria density dropped to 4000 +/- 1000 and 3E5 +/- 1E5 CFU/mL in S. epidermidis and S. aureus groups, respectively (P < 0.001, P = 0.030). In group 4, there was uniform bacterial growth (S. epidermidis, 1.51E7 +/- 1.5E6; S. aureus, 1.48E7 +/- 1.5E6) before and after treatment. Conclusions: TBO-mediated PACT had an antibacterial efficacy in vitro on S. epidermidis and S. aureus isolated from ocular surface infection. Translational Relevance: As TBO-mediated PACT has a strong antibacterial effect to S. epidermidis and S. aureus in vitro, this approach may assist in the treatment of ocular surface infectious diseases.