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A previous ethnobotanical investigation conducted in Valle Imagna (Northern Italy) highlighted the traditional use of Agrimonia eupatoria L. (Rosaceae) as a disinfectant and wound-healing agent. This use seemed to be linked to a local 18th century manuscript. This species was chosen for a multidisciplinary investigation to validate or refute its traditional use in the valley. Samples from fresh leaves were observed under Scanning Electron and Light Microscopy. The phenolic profiles of an epicuticular aqueous extract of the whole leaves and of infusions and decoctions of leaves and aerial parts were analyzed through Liquid Chromatography–Tandem Mass Spectrometry. The volatile organic compounds (VOCs) of fresh leaves were analyzed through Headspace Solid-Phase Microextraction coupled with Gas Chromatography–Mass Spectrometry. Growth inhibition and adhesion modulation were assessed on Escherichia coli, Staphylococcus aureus, and S. warneri by minimum inhibitory concentration and adhesion assays. Two trichome morphotypes were observed: a capitate with a one-celled rounded head and a capitate with a teo-celled cylindrical head. Both were responsible for producing terpenes, while the cylindrical capitates also produced polyphenols. Thirty-four phenolic compounds were characterized. Luteolin-7-O-glucoside, Catechin, and Epicatechin were common to all five extracts. The VOC profiles highlighted the dominance of (+)-α-Pinene. The infusions and the decoctions had a significant inhibitory activity on E. coli, and the extracts (specifically, the infusion of the leaves and both decoctions) also had a stimulating effect on the biofilm formation of S. warneri. These results already hold particular interest because of the strong connection they have to the traditional use of agrimony described in Valle Imagna. Full article