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The growing concern on the antibiotic resistance spreading among bacteria has stimulated the search for valuable alternatives from plant sources. This study dealt with the potential use of hemp (Cannabis sativa L.) seeds extract to inhibit the growth of selected pathogenic enterobacteria and the biofilm formation by Staphylococcus aureus , representing severe risks of food-borne illnesses. Effects on probiotic bacteria were also examined. A double-staining viability/mortality assay was used to examine potential S. aureus membrane damage. Our results highlighted a selective antimicrobial activity of C. sativa extract against pathogenic strains and no inhibitory effects on the growth of probiotic strains belonging to the Bifidobacterium and Lactobacillus genera. This selective inhibition is of outmost importance for the maintenance of healthy gut microbiota. The double-staining assay showed that the C. sativa extract was capable of inhibiting the biofilm producer S. aureus ATCC 35556 strain; this antibacterial action was only partially linked to membrane damage. Biofilm formation was inhibited as well; inhibition occurs at lower concentration with respect to planktonic cells (0.5 mg/ml vs 1 mg/ml, respectively). Therefore, hemp seeds extracts represent a new exploitable and valuable antimicrobial and antibiofilm agent for the food and nutraceutical industry as a possible alternative to antibiotics/antibacterial compounds. Image 1 • Cannabis sativa L. seeds showed antimicrobial and antibiofilm activity. • C. sativa L. seeds selectively inhibit the growth of potentially pathogenic strains. • C. sativa L. seeds did not exert antimicrobial activity against probiotic bacteria. • C. sativa L. seeds inhibit the biofilm formation by Staphylococcus aureus. [ABSTRACT FROM AUTHOR]