Antioxidant and antimicrobial activity of the ethyl acetate and water subfractions of methanolic extracts of three Spanish onion varieties were assayed. Flavonoids were mainly present in ethyl acetate subfraction being 34.92 ± 0.75, 7.95 ± 0.16, 0.38 ± 0.01 μmol of rutin eq. g−1 D.W. and its antioxidant capacity was 74.86 ± 1.77, 24.59 ± 0.67, 4.55 ± 0.44 μmol Trolox g−1 D.W. of Grano de Oro, Fuentes de Ebro and Calçot de Valls varieties, respectively. The antimicrobial activity of flavonol standards and onion extracts was evaluated against some food spoiler microorganisms. Quercetin and kaempferol were inhibitory against gram positive bacteria such as Bacillus cereus, Staphylococcus aureus, Microcroccus luteus and Listeria monocytogenes. Gram negative bacteria Escherichia coli and Pseudomonas aeruginosa were less sensible to the antimicrobial effect of both flavonol standards and Candida albicans was totally resistant. Among the onion extracts tested only ethyl acetate subfraction showed antimicrobial inhibition.