A large scale screening of the in vitro susceptibility of 105 strains of Prototheca zopfii to a panel of polyene antibiotics (amphotericin B, nystatin, primaricin and filipin) was conducted. Strains studied were isolated from dairy-associated environments in five different localities. Groups 1-4 included strains recovered from four separate regions of Italy, while group 5 included isolates from Belgium. Amphotericin B and primaricin exhibited the highest activity, with th MIC90 ranging from 4 and 8 microg/ml, respectively. On the other hand, the MIC90 of nystatin and filipin were from two to four times higher. Two strains were resistant to all four polyenes tested. The above results are compared with those in the literature and the importance of carrying out large-scale screening surveys to assess polyene susceptibility patterns within the species P. zopfii is discussed.