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Two outbreaks of mastitis due to the yeast-like alga Prototheca zopfii recently occurred in dairy herds in Lombardia (Italy) involving 180 and 150 lactating cows, respectively.To determine the in vitro susceptibility of Prototheca isolates to conventional antifungal agents and to essential oils.Twenty P. zopfii isolated from milk during these outbreaks, six P. zopfii isolated from fresh water and two Prototheca sp. reference strains were submitted to antifungal susceptibility testing by broth microdilution assay following the CLSI guidelines for yeasts.The tested isolates were shown to be resistant to fluconazole and caspofungin. A wide range of voriconazole MICs was observed. In contrast, amphotericin B, itraconazole and posaconazole appeared active with MICs ≤ 1 mg/L. Bergamot and tea tree oils seemed to exert an interesting activity against this yeast-like alga.Difficulties in treating animals with conventional drugs and the potent in vitro activity of essential oils demonstrated here raise the interest in further investigations on the therapeutic use of these non-conventional natural products.