The aim of this study was to clarify the seasonal variation in the proximate composition of the free amino acid (AA) and fatty acid (FA) profiles of the European sardine (Sardina pilchardus) from the Adriatic Sea and to better understand the nutritive value needed to organize more effective industrial processing, aquaculture use and to ensure the health benefits for consumers through available bioactive compounds such as omega-3 FA and essential AA. The lipid content ranged from 1.18 to 10.58% during the year, being the highest from July to September. For the first time, this paper reports the monthly variation in AA content in sardines. The highest total AA content was measured during the winter period, from January (843 mg/100 g fillet) to March (953 mg/100 g) with histidine, arginine and threonine being the most dominant. The total content of essential free AA (histidine, threonine, valine, methionine, isoleucine, leucine, phenylalanine, tryptophan and lysine) ranged from 137 to 571 mg/100 g fillet (wet weight), recorded in May and March, respectively. The fatty acid profile analyses revealed the major saturated FA as palmitic (C16:0), followed by myristic (C14:0), and stearic (C18:0) acids, and the predominant monosaturated FA as oleic (C18:1n–9) and palmitoleic (C16:1n–7). The high concentrations of polyunsaturated FA in sardines were omega-3 FA, particularly eicosapentaenoic (EPA; 20:5n-3) and docosahexaenoic (DHA; 20:6n-3) FA. From July to September, their content was the highest (>3.5 g/100 g of sardine fillets), confirming that these species are excellent sources of bioactive lipids.