Omega-3 long-chain polyunsaturated fatty acids (n-3 LC PUFAs) and vitamin D₃ are essential components of human nutrition. A regular human diet is highly deficient in n-3 LC PUFAs. Fish like salmon are highly recommended in the human diet as they are a major source of high-value n-3 LC PUFAs and vitamin D_3. The levels of these nutrients have been decreasing over the last few years in farmed salmon, whose production urgently needs sustainable sources of these nutrients. The microalga Nannochloropsis gaditana (NG) is known for its naturally high potential for the production of eicosapentaenoic (EPA, 20:5 n-3) fatty acid. A commercial diet for Atlantic salmon was supplemented with 1% and 10% of spray-dried NG grown under controlled conditions for a high EPA content. Salmon were harvested on day 49, following which, boneless and skinless salmon meat was recovered from fish and analyzed for the fatty acid profile, total fat, and vitamin D₃. Vitamin D₃, EPA, and docosapentaenoic fatty acid (DPA, 22:5 n-3) levels were significantly increased (p < 0.05) by supplementing the basal diet with 10% NG, thus, NG represents a novel, functional, natural ingredient and a sustainable source of n-3 LC-PUFAs that can raise the levels of healthy fats and vitamin D₃ in farmed salmon meat.