The antimicrobial activity of toluidine blue associated with red light has been demonstrated for a wide range of microorganisms including those commonly found in infected root canals, carious lesions and periodontal pockets. Recent reports have drawn attention to the problems of antimicrobial resistance and resistance of oral bacteria to antibiotics and local antiseptics is of increasing concern, thus photodynamic therapy could be an alternative antimicrobial approach to treat localized infections in oral cavity. In this study the fluorescence spectra of TB were obtained before and after laser exposure in the presence or absence of Streptococcus mutans. The dye concentration was 0.01%, the irradiation was performed with a diode laser, l= 660 nm, P= 40mW, exposure time of 3 minutes in a volume of 0.5 ml, with a pre-irradiation time (PIT) of one or five minutes. The results showed shifts in fluorescence spectra observed for different preirradiation times in the presence of S. mutans. In the absence of bacteria, a shift in the spectra was observed in the dye before and after irradiation. These findings may indicate a photobleaching of the dye denoting structural alterations after irradiation and confirm the importance of the PIT for the success of this therapy.