Members of the genus Prototheca are aerobic, unicellular algae related to the green algae of the genus Chlorella, but without chlorophyll. They are resistant and ubiquitous and can be isolated from a great variety of environmental sources. Many species of Prototheca genus have been identified but only two P. zopfii and P. wickerhamii, are able to produced infection in humans and animals. Frequently P. zopfii is most incriminated into the pathologic process in animals, and P. wickerhamii in humans, but is still hard to establish a strict delimitation. By different animal species, the most sensitive seems to be the cows, in witch will evolve as enzootic mastitis, and dogs in witch some systemic infections are described. Majority of humans infections are produced by P. wickerhamii and were mostly described in patients with immunosuppressant disorders, chronic diseases or after intensive treatments with various antibiotics. In this study we report a FISH method optimization, for Prototheca, which can be used for rapid diagnosis both in humans as well as in animals protothecosis. Reliable and quick identification of P. zopfii and P. wickerhamii is important for accurate treatment and understanding their role in the pathogenesis of infections. Fluorescent in situ hybridization (FISH) of Prototheca algae with specific eukaryotic probe EUK 516 ACCA GAC TTG CCC TCC leads to a reduced time to identification. In clinical practice, FISH therefore can be used in situations in which quick identification is necessary for optimal treatment of the patient.