AbstractFreezing is an efficient method of fish preservation. The aim of the present work was to examine the impact of freezing in fatty acid composition and in the in vitro inhibitory activity of sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) fillet lipids against platelet activating factor (PAF). The Bligh and Dyer extraction method and the counter‐current distribution method were used to obtain total, polar and neutral lipids. The fatty acid analysis conducted using the internal standard method and the biological assay on washed rabbit platelets took place calculating the in vitro inhibitory activity of fish lipids against 2.5 × 10−11 M of PAF. No statistical changes (p<0.05) occurred in fatty acid content of fresh and thawed gilthead sea bream, while fatty acid amount in thawed sea bass was significantly higher (p<0.05) compared to fresh fish. Total lipids of both thawed fish species exhibited stronger anti‐thrombotic activity compared to fresh fish. Freezing preserved fish quality and reinforced the anti‐thrombotic properties of fish oils, since even after 6 months of freezing, fish oils preserve their nutritional value in terms of protecting against cardiovascular diseases.Practical applications: Fish fillets contain high amount of unsaturated lipids that may easily undergo lipid oxidation. Freezing and frozen storage prevent such oxidative changes so fish quality is retained. Fatty acids and PAF‐antagonists in fish are of major importance since they contribute to the nutritional value of fish. The practical application of this work lies on the evaluation of the nutritional value of fish in terms of cardio protection by examining the impact of freezing on the levels of fatty acids and PAF‐antagonists in aquacultured fish fillets.