A lignan glycoside identified as lyoniside (9-O-β-d-xylopyranosyl(+)lyoniresinol) was obtained from ethanol extracts of the rhizomes and stems of bilberry (Vaccinium myrtillus L.), on a preparative scale, by droplet counter-current chromatography. The application of this method permitted the isolation of a pure substance in only one chromatographical step. The occurrence of lyoniside in bilberry is reported for the first time. Seasonal fluctuations in the content of this compound in plant organs were demonstrated showing its highest levels in bilberry rhizomes and stems during the winter and their subsequent decrease in the spring. In vitro, the purified lyoniside was evaluated for antioxidant, allelopathic and antifungal activities. It showed significant radical scavenging properties in a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay with IC50 of 23 μg ml−1. Applied at concentration of 10 μg ml−1, it suppressed by 75% the seedling radical growth of Lactuca sativa and Lepidium sativum, and exerted strong inhibitory effect (55%) on germination of Larix decidua. Moreover, the synergistic action of lyoniside and triterpene acids was demonstrated in inhibitory effect exerted on germination and growth of Pinus sylvestris. Among 5 tested fungi strains of Ascomycota, the highest susceptibility was shown by Fusarium oxysporum and Mucor hiemalis, with mycelial growth inhibited by lyoniside concentration of 50 μg ml−1 by 78 and 80%, respectively