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Onion (Allium cepaL.) is a very important vegetable crop cultivated worldwide. The bulb is a major source of phytochemicals useful for human health and rich in sulphur compounds responsible for their typical odour and flavour. In this study, we investigated the volatile compounds and biochemical properties besides the antimicrobial activity of onion Montoroand Alifevarieties, and of two ecotypes of Vatollavariety (spinning top and tapered shape), cropped in the southern Mediterranean area (Italy). For all investigated onions, the organosulphur compounds, mainly di- and trisulfides, are the most abundant compounds. Alifevariety showed higher polyphenols amount (8.2 GAE mg/g dw) with respect to the lowest one (3.9 GAE mg/dw g) in spinning top Vatolla, as well as an higher antioxidant activity (42.37 µmol TE/g dw) about two-fold higher than those detected in the other varieties. All the onions showed low pungency level, confirming their popular classification as a sweet onion. The total content of soluble sugars ranged from 461 to 624 mg/g dw; malic acid was the major organic acid in Alifeand Montorovarieties instead for both Vatollaecotypes citric acid was the most abundant ones. The biochemical characterization highlighted the three onion varieties as a good source of bioactive compounds. The antimicrobial activity of the onion extracts pointed out an effective action against three Gram-positive species (B. cereus, L. innocua, S. aureus) and P. aeruginosa; consequently, they could represent a new source of natural antimicrobial agents.