Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides brasiliensis. Currently, the treatment approach involves the use of antifungal drugs and requires years of medical therapy, which can induce nephrotoxicity and lead to resistance in yeast strains. Photodynamic inactivation (PDI) is a new therapy capable of killing microorganisms via the combination of a nontoxic dye with visible light to generate toxic reactive oxygen species (ROS). We investigated the phototoxic effect of 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin (TMPyP), a cationic porphyrin, on the survival of P. brasiliensis following exposure to light. Phototoxicity was found to depend on both the fluence and concentration of the photosensitizer (PS). Although the biological effects of PDI are known, the molecular mechanisms underlying the resultant damage to cells are poorly defined. Therefore, we evaluated the molecular response to PDI-induced oxidative stress by gene transcription analysis. We selected genes associated with the high-osmolarity glycerol (HOG)-mitogen-activated protein kinase (MAPK) pathway and antioxidant enzymes. The genes analyzed were all overexpressed after PDI treatment, suggesting that the oxidative stress generated in our experimental conditions induces antioxidant activity. In addition to PDI-induced gene expression, there was high cell mortality, suggesting that the antioxidant response was not sufficient to avoid fungal mortality.