The growing interest in functional foods has prompted the development of innovative bio-fortification strategies aimed at enhancing the nutritional profile of animal-derived products. This study presents a systematic analytical investigation into the bio-fortification of farmed Sparus aurata (sea bream) fillets through the application of a polyphenol-rich extract derived from artichoke by-products. The extract was obtained via ultrasound-assisted enzymatic extraction from a suspension of artichoke leaves and stems. The phenolic compounds present in cooked fortified fillets were characterized by using HPLC coupled to a DAD detector, which proved useful in assessing 11 phenolic compounds. Notably, the 11 compounds characterized, included: 7 hydroxycinnamic acid derivatives (caffeoylquinic acids), namely 1-O-, 3-O-, 4-O-, and 5-O-caffeoylquinic acid; and 1,3-, 1,5-, 3,4-di-O-caffeoylquinic acid; and 4 flavones, namely luteolin, apigenin, luteolin-7-O-glucoside, and luteolin-7-O-rutinoside. Quantitative HPLC-DAD analyses revealed significantly higher concentrations of chlorogenic acid in fortified samples (1.65 ± 0.12 mg·g⁻¹ d.w. in VI group; 1.02 ± 0.08 mg·g⁻¹ d.w. in SC group) compared to control (CTR) samples (0.12 ± 0.06 mg·g⁻¹ d.w.). Additional phenolics including luteolin derivatives, cynarin, and caffeic acid were also identified in cooked fortified fillets. The antioxidant activity, determined through the DPPH assay, increased significantly in fortified samples: 42.5% ± 3.2% (VI), 38.9% ± 2.8% (SC), vs. 19.6% ± 1.5% (CTR). Microbial shelf-life analysis demonstrated an extension of 4 days (VI) and 3 days (SC) in refrigerated raw fillets. The sensory evaluation indicated improved texture and flavor in SC fillets. These results underscore the analytical effectiveness of both fortification methods in enhancing the nutritional, functional, and sensory attributes of fish fillets, supporting their potential application in the development of targeted functional seafood products.