The Achillea genus is one of the widely used genera to treat various medical ailments. In this study, gas chromatography (GS) and Solid Phase Microextraction (SPME) were used to determine the essential oil composition of the A. millefolium L. Human skin fi broblasts (HSF) viability based on spectrophotometrical 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Neutral Red (NR) methods and morphological analysis was performed in in vitro cell culture. Free radical scavenging activity of ethanol, ethyl acetate and water extracts of A. millefolium L. was also measured. The total oil content in Achillea fl owers was analyzed only with gas chromatography/mass spectrometry (GC/MS) method. SPME-GC/MS technique revealed that Achillea fl owers liberate high amounts of monoterpenes (α-pinene; β-pinene; 1,8-cineole). However, proportionally, most of all sesquiterpenes (β-caryophyllene and germacrene D) were found. We also showed that fresh fl owers contain much more monoterpenes such as α-pinene or sabinene but fewer amounts of β-pinene than preserved material. MTT analysis indicated that methanol extract from fresh, not dried, plant material and water extract from fresh plants at doses ranging from 25-225 μg/mL had no cytotoxic activity on HSF cells. Methanol extract from dried and freeze herb (doses >75 μg/mL) and ethyl acetate extract from fresh plants (doses >25 μg/mL) inhibited succinyl dehydrogenase activity. In the NR assay, only methanol extract from fresh plant material had no cytotoxic activity. The remainder signifi cantly decreased dye uptake. Moreover, ethyl acetate extract strongly infl uenced cellular actin fi laments composition, and consequently morphology of cells. Analysis of the IC50 values revealed that for methanol from fresh herb and water extracts, the IC50 values are higher than 250 μg/mL. The lowest IC50 was 13 μg/mL, obtained in NR assay for ethyl acetate extract. The highest free radical scavenging activity (1,1-diphenyl-2-picrylhydrazyl (DPPH) method) was found for methanol extract from dried herb at the maximal dose used (175 μg/mL), and was 19.2% higher than control value. When compared to the Trolox standard activity it was equivalent of the 6.8 μg/mL. In conclusion, the results suggest that A. millefolium L. extracts may, in limited concentrations, be useful in preparations of topical application and considered for use in clinical dermatology.