This article reports the results concerning the qualitative and quantitative composition of the volatile oil obtained from the aerial parts of Tanacetum vulgare, collected in Lithuania, by supercritical fluid extraction (SFE) with CO2 at 90 bar and 40 °C and by hydrodistillation (HD). GC-MS and GC-FID analysis revealed that the oils mainly consisted of 1,8-cineole, trans-thujone, cis-chrysanthenol, borneol and myrtenol. Oils obtained by HD displayed, with respect to the SFE volatile oil, a higher amount of hydrocarbon monoterpenes. The hydrodistilled oil was particularly active against dermatophyte strains and Cryptococcus neoformans, with MIC values in the range of 0.16-0.64 μL/mL. The oil revealed a significant inhibitory effect on germ tube formation in Candida albicans at sub-inhibitory concentrations. At the concentration of 1/16 MIC, the inhibition of filamentation was more than 80% in comparison to untreated control cells.