| Summary: | <i>Epilobium angustifolium</i> L. is applied as an antiseptic agent in the treatment of skin diseases. However, there is a lack of information on human skin penetration of active ingredients with antioxidative potential. It seems crucial because bacterial infections of skin and subcutaneous tissue are common and partly depend on oxidative stress. Therefore, we evaluated in vitro human skin penetration of fireweed ethanol-water extracts (FEEs) by determining antioxidant activity of these extracts before and after penetration study using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and Folin–Ciocalteu methods. Microbiological tests of extracts were done. The qualitative and quantitative evaluation was performed using gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC-UV) methods. The in vitro human skin penetration using the Franz diffusion chamber was assessed. The high antioxidant activity of FEEs was found. Gallic acid (GA), chlorogenic acid (ChA), 3,4-dihydroxybenzoic acid (3,4-DHB), 4-hydroxybenzoic acid (4-HB), and caffeic acid (CA) were identified in the extracts. The antibacterial activities were found against <i>Serratia lutea</i>, <i>S. marcescens</i>, <i>Bacillus subtilis</i>, <i>B. pseudomycoides</i>, and <i>B. thuringiensis</i> and next <i>Enterococcus faecalis</i>, <i>E. faecium</i>, <i>Streptococcus pneumoniae</i>, <i>Pseudomonas aeruginosa</i>, and <i>P. fluorescens</i> strains. In vitro penetration studies showed the penetration of some phenolic acids and their accumulation in the skin. Our results confirm the importance of skin penetration studies to guarantee the efficacy of formulations containing <i>E. angustifolium</i> extracts.
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