Photodynamic therapy (PDT) has been originally developed for cancer treatment, but recently, it has been successfully employed against microorganisms, including fungi. Chromoblastomycosis is a subcutaneous fungal infection that is recalcitrant to conventional antifungal drug therapy. The most frequent species involved are Foncecaea pedrosoi and Cladophialophora carrionii. The present study aimed to verify the efficacy in vitro of PDT employing methylene blue (MB) as a photosensitiser and Light emmiting diode (LED) (InGaAl) as the light source. Methylene blue at the concentrations of 16, 32 and 64 μg/mL and LED (InGalP) were employed for 15 min against spores of two isolates of F. pedrosoi and two isolates of C. carrionii. The spores were plated on Sabouraud Dextrose agar and the number of colony forming units was counted after 7–10 days of incubation at 37 °C. The PDT with MB and LED was efficient in reducing the growth of all samples tested. Better results were obtained for the concentration of 32 μg/mL of MB. The treatment proved to be highly effective in killing the samples of F. pedrosoi and Cladophialophora pedrosoi tested in vitro. PDT arises as a promising alternative for the treatment of this subcutaneous infection.