The objective of this study was to evaluate the bactericidal effect of photodynamic antimicrobial chemotherapy (PACT) using phenothiazinium dye (Toluidine blue O and methylene blue) at a low concentration of 1μg/mL irradiated with the red laser at doses of 2.4 e 4.8 J/cm² on strain of Staphylococcus aureus (ATCC 23529) in vitro. For this research, tests were performed in triplicate and the samples were distributed into six test groups: (L-P-) Negative control (L1+P-) and (L2+P-) bacterial suspensions were irradiated with laser energy 2.4 and 4.8 J/cm2 respectively in the absence of photosensitizer; (L1+P+) and (L2+P+) bacterial suspensions were irradiated with laser in the presence of 1μg/ml of photosensitizer and finally (L-P+) bacterial suspensions only in the presence of phenothiazinium dye. Therefore, were analyzed the potential bactericidal PACT by counting of colony-forming units and analyzed statistically (ANOVA, Tukey test, p<0.05). The results showed that the negative control group when compared with laser group (L2+P-) it was observed a statistically significant increase (p<0.01) which L2+P- showed a higher number of CFU, on the other hand when compared to L1+P- no statistically significant difference was found, relation to the groups submitted to PACT, only showed a statistically significant reduction relative to the group irradiated L2+P+ (p<0.01) that showed a decrease in the number of CFU. There was no statistically significant difference between the groups submitted to PDT (L1+P+ and L2+P+). Although the results of this study have shown a reduction in average number of colony forming units by the appropriate laser-dye treatment combination, it needs further investigation.