This paper presents the results of the study of the composition of the essential oil (EO) of Artemisia rutifolia by the GC/MS method as well as its antimicrobial and antiradical activities. According to the PCA-analysis, these EOs can be conditionally divided into “Tajik” and “Buryat-Mongol” chemotypes. The first chemotype is characterized by the prevalence of α- and β-thujone, and the second chemotype by the prevalence of 4-phenyl-2-butanone, camphor. The greatest antimicrobial activity of A. rutifolia EO was observed against Gram-positive bacteria and fungi. The EO showed high antiradical activity with an IC50 value of 17.55 μL/mL. The presented first data on the composition and activity of the EO of A. rutifolia of the Russian flora indicate the prospects of the species as a raw material for the pharmaceutical and cosmetic industry.