The aim of the present work was to study the effect of a broccoli phytochemical extract (Br-ex) on the release of fatty acids (FA) from salmon muscle (SM) and salmon oil (SO) during in vitro digestion. The hypothesis of the study was that Br-ex contains polyphenols which might act as pancreatic lipase inhibitors. The effect on the release of specific FA, in particular the long-chain n-3 polyunsaturated fatty acids (PUFAs), EPA (C20:5 n-3) and DHA (C22:6 n-3), was recorded, and the impact of the SM matrix was studied by comparing the release of FA from SM and SO. In vitro digestion was performed and lipolytic activity, measured as the release of fatty acids (FFA) by solid phase extraction and GC-FID, was recorded at 20, 40, 80 and 140 minutes in the intestinal phase. The results showed, unexpectedly, that Br-ex stimulated the release of FA during digestion of SO and SM, showing the highest increases in FFA, 67% and 64%, respectively, at 20 min. No difference in the release of FA from SO compared to SM was observed, suggesting that the SM matrix had minor influence on the lipolytic activity. The results also demonstrated that the increase in lipolytic activity caused by Br-ex was not affected by the SM matrix. However, addition of Br-ex resulted in a lower percentage of EPA and DHA in the FFA fraction, suggesting that the lipase sn-position preference was altered. Whether this affects the bioaccessibility of EPA and DHA needs further investigation.