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Background:Plantago lanceolata L. (P. lanceolate) and Plantago major L. (P. major) belong to the Plantaginaceae family and are widely used in traditional medicine. Objectives: This study aims to qualitatively identify the crucial compounds and evaluate the toxicity effects of essential oils of two Plantago species. Methods: The plantains were collected from Zanjan Province, Iran. The essential oils were extracted by hydrodistillation and then analyzed using gas chromatography coupled with mass spectrometry (GC/MS). The toxicity effects of the essential oils were evaluated on HCT-116 and HEK-293 cell lines (in vitro MTT assay) and Artemia salina (A.salina) (in vivo assay). The constituents of the essential oils were identified by calculating their retention indices under temperature-programmed conditions for n-alkanes (C8 -C20) in the Agilent 19091S-433 column. Results: The main identified constituents were metaraminol (14.04%), bifemelane (8.73%), metossamina (8.16%), and pterin-6-carboxylic acid (5.11%) in P. lanceolata and 2-dodecen-1-yl (-) succinic anhydride (15.29%), benzenemethanol, α-(1-aminoethyl)-2,5- dimethoxy-(11.83%), dl-phenylephrine (7.51%), and nortriptyline (5.15%) in P. major. The essential oils of P. major exhibited more antiproliferative properties on HCT-116 at 72 h compared to P. lanceolata (IC50: 102.66 µg/mL). At 400 µg/mL of P. lanceolata and P. major, the percentage of the lethality of nauplii was 8% and 12%, respectively (LC50:2242.57 µg/ mL and 1783.7 µg/mL). The present study showed that the most of constituents of oils were alcohols and amines. Conclusion: Some of the compounds identified in the Plantago species essential oils have important pharmaceutical properties. This study reported the cytotoxicity of essential oils on the colon cancer cell line. However, the essential oils were not toxic against A.salina at the examined concentrations.