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Plants contain bioactive substances and secondary metabolites that have a variety of functions, including antibacterial, antioxidant, anti-inflammatory, and anticancer activities. In this study, the content of bioactive compounds in five medicinal plants was determined, i.e., Plantago major L., Armoracia rusticana, Hypericum perforatum L., Rumex acetosa L., and Urtica dioica L., from 38 locations. Additionally, the antimicrobial effect of extracts of bioactive compounds from the above-mentioned plants was checked. The experiment used an original method of extracting bioactive compounds. Purpose of the research: the assessment of antimicrobial activity and chemical characterization of extracts obtained using our own method of isolating bioactive compounds from green parts of medical plants in Poland. Based on the research, the presence of bioactive compounds, i.e., phenolic acids and flavonoids, was found in the tested plant extracts. The results of this study suggest that the geographic parameters of the locations where these plants grow have different effects on their biochemical composition and biological activity. The results showed that all tested plants had significant antibacterial activities. Rumex acetose L. showed the highest antimicrobial activity against Escherichia coli and Salmonella enteritidis. These studies supplement the existing literature on the subject with information about the antimicrobial properties of the tested plant extracts that can be used in herbal medicine. The results have significant implications for the pharmaceutical, nutraceutical, and cosmetic sectors, establishing a foundation for future research in this area. [ABSTRACT FROM AUTHOR]