Echinocandins are antifungal drugs used for the treatment of invasive candidiasis and aspergillosis. They bind to serum proteins within a rate of 96 to >99%. The effect of serum on in vitro echinocandin susceptibility tests of certain Candida and Aspergillus species was reported. This study was performed to determine the effect of human serum on in vitro susceptibility testing of echinocandins for clinical isolates of Candida parapsilosis and Candida guilliermondii, the species which generally have higher minimum inhibitor concentrations compared with other Candida species. One hundred C. parapsilosis and 20 C. guilliermondii isolates were included in the study. The susceptibility tests of caspofungin, micafungin and anidulafungin were performed using microdilution method, either in the presence or absence of 50% human serum, according to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines. It was demonstrated that human serum significantly affects the in vitro susceptibility results of echinocandins for C. parapsilosis and C. guilliermondii isolates, mostly yielding an increase in MICs. The most prominent fold changes were for micafungin and anidulafungin in C. parapsilosis, and for anidulafungin in C. guilliermondii isolates. Serum influences the in vitro echinocandin susceptibility in C. parapsilosis and C. guilliermondii. The mechanism and clinical significance of this in vitro change need to be clarified.