This study comprehensively characterised a protected designation of origin mussel ‘Cozza di Scardovari’ (Mytilus galloprovincialis) by examining how it is affected by the farming site (outer vs. inner area of the lagoon) and harvest time (21 April vs. 18 May vs. 16 June). Harvest time affected the marketable traits and fatty acid profile of mussels, whereas farming site scarcely affected marketable traits and mussel yields. Mussels from the inner area of the lagoon displayed a superior nutritional profile, including higher contents of proteins (7.8% vs. 7.4%; P < 0.05), lipids (1.2% vs. 1.0%; P < 0.001) and essential amino acids such as tryptophan (+24%; P < 0.05) and valine (+8%; P < 0.05), with a more favourable n-3/n-6 ratio (7.7 vs. 7.0; P < 0.001) than those from the close-to-sea area. Volatile organic compounds, mainly octanoic acid, dimethyl sulphide and 1-penten-3-ol, differed between farming sites within the same lagoon. •This is the first study to comprehensively characterise a Mediterranean mussel.•‘Cozza di Scardovari’ mussels were remarkably low in sodium and heavy metals.•The lagoon environment influenced mussel nutritional and volatile profiles.•Harvest time largely affected the marketable traits and fatty acids profile of mussels.