In this work, we evaluated the effects of laser treatment on fungal cells, aiming to provide support for the use of this therapy during Paracoccidioidomycosis (PCM). HeNe laser treatment reduced the growth capacity and the viability of fungal cells, and increased gp43 expression. Laser-treated cells also showed pronounced structural alterations, since they were collapsed and presented deep folds. Additionally, remarkable changes were observed in the nucleus that presented a very electron dense form. The pathogenicity of P. brasiliensis after laser treatment was also evaluated, and no differences were observed in pulmonary lesions, comparing animals infected with laser-treated and non-treated yeast cells. Possibly HeNe laser is able to modify fungal structures involved in resistance and virulence mechanisms and therefore can be considered as an adjunctive therapy in the treatment of skin lesions observed during PCM.