Global population growth, climate change, and the reduced resilience capacity of ecosystems now require an increased use of renewable biological resources. Fish consumption and the popularity of seafood are rising due to their undeniable benefits for human health; however, this has led to an increase in the production of fishprocessing waste. Most of this waste ends up in the sea or in landfills and poor valorized for the production of feed, causing not only disposal problems with negative environmental impacts, but also significant economic losses. From this perspective, the valorization of fish-processing waste through biotechnological procedures to produce high value-added compounds gives the bioeconomy a strategic role within the framework of the circular economy. Fish-processing by-products include a substantial amount of solid waste such as heads, bones, viscera, skin, fins, blood, intestines, and underutilized low-value fish, which are rich in nutrients, including proteins (essential amino acids, peptides), carotenoids, lipids, and long-chain omega-3 polyunsaturated fatty acids (PUFAs), polysaccharides (chitosan, chitin, glycosaminoglycans), vitamins, and minerals.This study aims to characterize fish viscera to analyze the high value-added compounds present in these by-products. Supercritical carbon dioxide extraction (CO₂–SFE) was investigated as an eco-friendly and safe technology for defatting the matrices, by modifying temperature, pressure, and time parameters. CO₂–SFE was tested at 350 bar and a temperature of 60 °C, with a solvent-to-biomass ratio ranging from 0.6 to 1.0 kg of CO₂ per kg of biomass. After extraction, the extracts and matrices were characterized to assess the potential use of the extracted compounds for human nutrition. Biomolecules of fundamental importance, such as proteins, carbohydrates, and lipids, were detected. The results highlighted that CO2-SFE can be employed as an efficient technology to obtain biomolecules of interest from fish-processing waste.