Bovine mastitis caused by Prototheca spp. can be a disease of high significance because of economic losses and the potential risk to public health. The aim of our study was to evaluate enzymatic activity of Prototheca zopfii. For this study, we used 15 P. zopfii strains previously isolated from cows with clinical and subclinical mastitis in Poland. We determined enzymatic profile of Prototheca species using the API ZYM system. Of the enzymatic activities detected during the study, acid phosphatase, leucine arylamidase, naphthol-as-bi-phosphohydrolase, esterase, lipase esterase, valine arylamidase, alkaline phosphatase, and lipase C14 were found in high percentage of strains.