The chemical composition, antioxidant potential and antimicrobial activity were studied in six walnuts (Juglans regia L.) cultivars (cv. Franquette, Lara, Marbot, Mayette, Mellanaise and Parisienne) produced in Portugal. Concerning their chemical composition the main constituent of fruits was fat ranging from 78.83% to 82.14%, being the nutritional value around 720kcal per 100g of fruits. Linoleic acid was the major fatty acid reaching the maximum value of 60.30% (cv. Lara) followed by oleic, linolenic and palmitic acids. The aqueous extracts of walnut cultivars were investigated by the reducing power assay, the scavenging effect on DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals and beta-carotene linoleate model system. All the walnut extracts exhibited antioxidant capacity in a concentration-dependent manner being the lowest EC(50) values obtained with extracts of cv. Parisienne. Their antimicrobial capacity was also checked against gram positive (Bacillus cereus, Bacillus subtilis, Staphylococcus aureus) and gram negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae) and fungi (Candida albicans, Cryptococcus neoformans), revealing activity against the different tested microorganisms.