© 2019 by the authors. The in vitro antithrombotic properties of polar lipid constituents of malted grain (MG), pelleted hops (PH), brewer's spent grain (BSG), spent hops (SH), wort, and bottled beer from the same production line were assessed in human platelets. The total lipids (TL) were extracted according to the Bligh and Dyer method and further separated into the total neutral lipids (TNL) and total polar lipids (TPL) extracts by counter-current distribution. The TL, TNL, and TPL extracts of all samples were assessed for their ability to inhibit platelet-activating factor (PAF) and thrombin-induced human platelet aggregation. The raw materials, by-products, wort, and beer lipid extracts all exhibited antithrombotic properties against PAF and thrombin. However, the beer TPL exhibited the lowest IC50 values against PAF-induced (7.8 ± 3.9 ¿g) and thrombin-induced (4.3 ± 3.0 ¿g) platelet aggregation indicating that these polar lipids were the most antithrombotic. The lipid extracts tended to be more bioactive against the thrombin pathway. The fatty acid content of all the TPL extracts were assessed using GC-MS. The fatty acid composition of the most bioactive TPL extracts, the wort and the beer, shared similar fatty acid profiles. Indeed, it was noted that fermentation seems to play a role in increasing the antithrombotic properties of polar lipids against PAF and thrombin by moderately altering the polar lipid fatty acid composition. Furthermore, the use of brewing by-products as a source of functional cardioprotective lipids warrants further investigation and valorisation.