Candida albicans is an opportunistic fungal species able to produce both superficial and systemic infections in immunocompromised patients. It has been demonstrated that biofilms produced by C. albicans are resistant to various antifungal drugs, and that the infections related to biofilm formation are often refractory to the conventional treatments. Photodynamic antimicrobial chemotherapy (PACT) is a potential antimicrobial therapy, which combines visible light and a non-toxic dye known as a photosensitizer, producing reactive oxygen species (ROS), which can kill the treated cells. In this work, we investigate the effects of PACT, using Toluidine blue (TB) as a photosensitizer, on 24-hour-old biofilms produced by C. albicans. It was observed that PACT using TB was able to decrease the viability of the 24-hour-old biofilms produced, in a TB concentration-dependent manner. The inhibition promoted by PACT (0.1 mg/ml TB) was in the order of 30%, 40% and 50% in the 24-hour-old biofilms submitted to incubation times of 1, 2 and 3 hours after PACT, respectively. At the same time, an increase in the ROS production was observed after PACT. Our results suggest that the inhibition observed in the viability of the 24-hour-old biofilms, by PACT, could be related to the increase in ROS production, which increases the cell permeability and leads to the damage of biofilms produced by C. albicans.