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In this work, the phytochemical composition and the biological activity of the ethanolic extracts obtained from Ajuga reptans L. (Lamiaceae) leaves and roots (growing in Lesser Poland Voivodeship, Poland) were compared. The phytochemical composition of the extracts were determined by the high-performance liquid chromatography with diode-array detection (HPLC–DAD) method. The dominant compounds in both extracts were verbacoside, isoverbacoside, 3,4-dihydroxyphenylacetic acid and rosmarinic acid. The antioxidant capacity of the extracts was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP) and 2,2’-azobis(3-ethylbenzothiazoline-6-sulfonate (ABTS) tests. The Folin–Ciocalteu assay was used to determine the total polyphenolic content. Additionally, enzyme (tyrosinase, collagenase) inhibition tests and metal chelating ability were studied to assess the anti-aging properties of the extracts. Moreover, the A. reptans extracts’ capacity to absorb the whole range of ultraviolet radiation and high-energy visible (HEV) light was evaluated. The skin irritation test (SIT) EpiDerm was applied to evaluate the safety of the bugle extracts. The noteworthy point is that there is a lack of literature on the assessment of A. reptans root extract activity. Our study is the only one that compares the quantitative composition and biological activity of extracts from the root and leaves of A. reptans. The obtained results indicated that both of the extracts exhibit high antioxidant, chelating and photoprotective activity, but the extract from A. reptans roots showed a higher enzyme inhibition effect for mature skin. The A. reptans root extract, similarly to the leaf extract, could be applied as potentially multi-functional, safe and sensitive cosmetic raw materials, especially in anti-aging and anti-pollution cosmetics. Full article