Background:Tanacetum parthenium(L.) Sch.Bip. (T. parthenium) is an aromatic perennial plant belonging to the Asteraceae family, also known as feverfew. It is widely distributed in various regions of Europe and other parts of the world. The plant has a rich background in the traditional medicine of many nations and has been used as a remedy for fever, pain, inflammation, asthma, rheumatism, menstrual disorders, etc. Methods: GC–MS analysis was conducted to determine the chemical composition of the isolated essential oil (EO). Using the method proposed by Litchfield and Wilcoxon, the average lethal dose (LD50) of the EO on Wistar rats was determined for two routes of administration: oral (p.o.) and intraperitoneal (i.p.). The subacute toxicity of the EO was also tested by oral administration of a daily dose of 1.0 g/kg body weight (BW) for 28 days. The toxicity of the EO was evaluated by observing and evaluating changes in behavior, body weight, basic hematological and serum biochemical parameters, and histopathological changes of the internal organs. Results: Thirty-seven volatile organic compounds representing 94.58% of the total oil composition were tentatively detected in the obtainedT. partheniumEO. The dominant compounds were camphor (45.47%),trans-chrisantenyl acetate (21.65%), camphene (9.48%), andcis-isogeraniol (5.42%). The results showed that the EO was not toxic when administered in acute oral doses. The acute mean lethal dose for intraperitoneal administration was LD50i.p. = 2.13 g/kg BW. In the subacute study involving administration of an oral dose of EO for 28 days, there were a number of changes in the hematological and serum biochemical parameters of the blood compared with the control group of animals. However, no symptoms of toxicity, changes in the body weight of the rats, death, or pathological changes in the histological indicators of the examined organs—brain, heart, stomach, liver, spleen and kidney—were found. Extrapolating the results obtained from the rat experiments, we can state that the EO is safe for use in doses below 1 g/kgBW for a period not exceeding one month.