Eggs of the common barbel (Barbus barbus) cause intoxication in humans after ingestion. In this work, the chemical composition of the haemolytically active fraction from methanolic barbel roe extract was analyzed. Compounds showing haemolytic activity and cytotoxicity towards normal and transformed cell lines were isolated and identified as polyunsaturated fatty acids, using online liquid chromatography–electrospray ionization mass spectrometry through tandem fragmentation experiments (HPLC–MS/MS). Arachidonic acid (C20:4), docosahexaenoic acid (C22:6) and eicosapentaenoic acid (C20:5) proved to be the three most abundant members of a complex series of free fatty acids ranging from C14:0 to C24:5. ► Eggs of the common barbel (Barbus barbus) cause intoxication. ► Barbel eggs contain haemolytic and cytotoxic polyunsaturated fatty acids. ► Polyunsaturated fatty acids were analysed by mass spectrometry. ► Arachidonic, docosahexaenoic, and eicosapentaenoic acids are the three most abundant members. ► Polyunsaturated fatty acids are responsible for the observed toxicity of barbel eggs.