Staphylococci are important causes of nosocomial and medical-device-related infections. Their virulence is attributed to the elaboration of biofilms that protect the organisms from immune system clearance and to increased resistance to phagocytosis and antibiotics. Photodynamic treatment (PDT) has been proposed as an alternative approach for the inactivation of bacteria in biofilms. In this study, we have investigated the effect of the photodynamic action of toluidine blue O (TBO) on the viability and structure of biofilms of Staphylococcus epidermidis and of a methicillin-resistant Staphylococcus aureus strain. Significant inactivation of cells was observed when staphylococcal biofilms were exposed to TBO and laser simultaneously. The effect was found to be light dose dependent. Confocal laser scanning microscopic study suggested damage to bacterial cell membranes in photodynamically treated biofilms. In addition, scanning electron microscopy provided direct evidence for the disruption of biofilm structure and a decrease in cell numbers in photodynamically treated biofilms. Furthermore, the treatment of biofilms with tetrasodium EDTA followed by PDT enhanced the photodynamic efficacy of TBO in S. epidermidis, but not in S. aureus, biofilms. The results suggest that photodynamic treatment may be a useful approach for the inactivation of staphylococcal biofilms adhering to solid surfaces of medical implants.