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Stachys lavandulifolia Vahl is a herbaceous plant distributed in the west and south western Asia. Despite of the wide medicinal uses, there are some reports on toxicity potential of this plant. In present study we attempted to evaluate the toxicity and to characterize the cytotoxic principles of S. lavandulifolia. Brine shrimp lethality test (BSLT) was used to evaluate the general toxicity of the extracts and essential oil obtained from the aerial parts of S. lavandulifolia. Phytochemical constituents of the active extract were investigated using various chromatographic and spectroscopic methods. GC and GC-MS were also used to analyze the plant essential oil. GC and GC-MS were used to analyze the plant essential oil. Cytotoxic activities of the isolated compounds were also evaluated using MTT assay method. In brine shrimp lethality test (BSLT), chloroform extract and the plant essential oil exhibited the most toxicity against Artemia salina larvae (LD50: 121.8±5.6 and 127.6±14.7 µg ml-1, respectively). GC and GC-MS analyses of essential oil led to the characterization of forty compounds of which α-bisabolol (23.85%) and thymol (17.88%) were identified as the main constituents. Nine flavonoids, Pachypodol (1), chrysosplenetin (2), kumatakenin (3), velutin (4), penduletin (5), viscosine (6), chrysoeriol (7), hydroxygenkwanin (8) and apigenin (9) were isolated from the chloroform extract of S. lavandulifolia. Among the isolated compounds, chrysosplenetin (2), a polymethoxylated flavonoid, was found as the most toxic compound toward MDA-MB-23 and HT-29 cells, with IC50 values of 88.23 and 116.50 µg ml-1, respectively …