Candida spp. can cause mild-to-severe human infections. Certain species have been described as the etiologic agent of human mastitis, inflammation of the breast tissue. Mastitis affects millions of lactating women and can be a source of disease transmission to the infant. In this work, we report the detection of the unusual etiologic agent of human mastitis, Candida guilliermondii, isolated from the milk of a puerperal woman with subacute mastitis in Rio de Janeiro, Brazil. Species identification was performed by MALDI-TOF MS and genetic sequencing. The patient had a full recovery after antifungal therapy.