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Ambrosia artemisiifolia L. (common ragweed) is an invasive weed, which is well known for the strong allergenic effect of its pollen as well as for its invasiveness and impact in crop fields (e.g. causing yield losses). This species produces a broad range of sesquiterpenoids. In recent years, new bioactive molecules have been discovered in this plant, e.g. isabelin, a sesquiterpene dilactone. The bioactivity of isabelin has been already demonstrated on allergy-related receptors and its inhibitory effect on seeds of various plant species. Isabelin was tested for potential antimicrobial effects by using a selection of soil-borne bacteria and fungi and three human pathogens as model organisms. For the majority of microorganisms tested, no antimicrobial activity of isabelin was observed. However, isabelin revealed strong antimicrobial activity against the Gram-positive soil bacterium Paenibacillus sp. and against the Gram-positive, multidrug-resistant Staphylococcus aureus. The observed inhibitory activity of isabelin can enlighten the importance to study similar compounds for their effect on human pathogens and on soil and rhizosphere microorganisms.