Prototheca zopfii causes bovine mastitis that leads to reduced milk production. Since P. zopfii isolates from mastitis have been assigned P. zopfii genotype 2, it suggests that this genotype is the etiologic agent of the infection. However, isolates of P. zopfii have not been investigated with regard to their in vitro drug susceptibility. In this study, we examine the susceptibility of genotype 2 strains from bovine mastitis and genotype 1 isolates recovered from cow-barn surroundings. The in vitro susceptibility of ten isolates and the type strain (SAG2063(T)) of P. zopfii genotype 1, and equal number of genotype 2 isolates and the type strain (SAG2021(T)) were assessed by E-test against amphotericin B (AMB), gentamicin (GM), kanamycin (KM) and itraconazole (ITZ). Results showed that P. zopfii genotype 1 isolates are more susceptible in vitro to AMB, GM and KM than those of genotype 2. Moreover, genotype 2 isolates and seven isolates of genotype 1, including the type strain, are not susceptible to ITZ (>10 μg/ml). Thus, drug susceptibility of P. zopfii differs between these two genotypes.