The essential oil extracted from Tanacetum parthenium (L.) Sch.Bip. leaves, cultivated in Dalat, Vietnam, were analyzed using GC-MS. The analysis identified twenty-three constituents, making up 98.8% of the total oil. The primary components were trans-chrysanthenyl acetate (33.3%), camphor (19.2%), sesquisabinene (16.8%), germacrene D (8.0%), and bornyl angelate (4.2%). Subsequent bioassays were conducted to assess the biological activity of the essential oil. Using the agar well diffusion method, the essential oil demonstrated antimicrobial activity against all tested microorganisms, including four bacterial strains and one fungal strain. The inhibition zones ranged from 9.16 ± 1.25 mm to 21.83 ± 1.25 mm. In addition, cytotoxicity assays were performed on KB, HepG2, MCF-7, and A549 cancer cell lines using the MTT method. The essential oil demonstrated notable cytotoxic activity across all four cell lines, with IC50 values ranging from 62.12 ± 2.66 to 103.62 ± 4.31 µg/mL.