This study was carried out with the aim of determining the antifungal effect of the essential oil isolated from Achillea millefolium subsp. millefolium plant against pathogenic fungi. In order to test the antifungal effect of the oil, an analysis was conducted on a total of 4 pathogen fungi which included Candida albicans, Candida tropicalis, Candida parapsilosis and Saccharomyces cerevisiae, and the effect of the essential oil on the growth of these fungi was investigated. The essential oil of A. millefolium ssp. millefolium had varying degrees of effect on the tested fungi. The highest antifungal effect was found against S. cerevisiae; whereas the lowest antifungal effect was found against C. parapsilosis. Nystatin showed a higher activity than the essential oil of A. millefolium subsp. millefolium against the tested fungi. MIC values of the essential oil against the tested fungi ranged from 1.25 μl/ml to 10 μl/ml. The results obtained indicate that essential oil of A. millefolium subsp. millefolium can be used as an alternative to antifungal agents such as amphotericin, ketoconazole, and fluconazole.