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Wine production is one of the most important agricultural activities. The winemaking process generates a considerable volume of different residues characterized as by-products, such as pomace, seeds, stems, and skins. By-products are rich in polyphenols with antioxidant and antibacterial properties and may act as bacteriostatic or bactericidal agents against food-borne pathogens, improving food safety by enhancing antibiotic efficacy and reducing bacterial resistance. The aim of this study was to evaluate the phenolic composition and antioxidant activity of grape components (skins, seeds, and stems) from three red grape varieties (Periquita, Gamay, and Donzelinho Tinto) and determine their antibacterial activity against antibiotic-resistant bacteria, including Escherichia coli in food-producing animals and Listeria monocytogenes from food products and food-related environments. Ten phenolic compounds were quantified in these red grape varieties, with specific compounds found in different parts of the grape, including phenolic acids and flavonoids. Flavonoids are abundant in seeds and stems, malvidin-3-O-glucoside being the main anthocyanin in skins. The ethanolic extract from the seeds showed in vitro concentration-dependent activity against reactive species like •NO and O2•−. Gamay extract was the most effective, followed by Donzelinho Tinto and Periquita. Extracts showed varying antibacterial activity against Gram-positive and Gram-negative bacteria, with stronger effects on Gram-positive bacteria. L. monocytogenes was more susceptible, while E. coli was limited to three strains. Seeds exhibited the strongest antibacterial activity, followed by stems. The results of our study provide evidence of the potential of grape by-products, particularly seeds, as sources of bioactive compounds with antioxidant and antibacterial properties, offering promising avenues for enhancing food safety and combating antibiotic resistance in food production and related environments. [ABSTRACT FROM AUTHOR]