Platelets exert an essential role in vascular inflammation and thrombosis. Flavonoids are natural compounds employed for the clinical management of vascular disorders preventing capillary permeability, working as phlebotonics and improving the blood rheology, although their mechanism of action remains partially unknown. The effects of quercetin, rutin, diosmetin and diosmin were investigated in platelet activation utilizing blood from healthy and non-treated volunteers. The arrangement of the different activation states of platelets and GPIIb/IIIa receptor occupation was computed by flow cytometry working with calcium ionophore as pro-aggregant to provoke platelet activation and aggregation. The flavonoids studied demonstrated relevant antiplatelet activity through the blocked of GPIIb/IIIa receptors, the suppression of the platelet activation, as well as the pro-aggregate effect of calcium ionophore. Therefore, whichever of the active ingredients examined could be beneficious in the prevention of cardiovascular disease and this article also contributes to elucidate a new mechanism of action for these drugs. Display omitted •Platelets play a critical role in vascular inflammation and thrombosis.•Platelet-derived microparticles also exert pro-aggregant activity.•Flavonoids exhibit binding to the GPIIb/IIIa platelet receptor.•The pro-aggregant activity of calcium ionophore decreased in the presence of flavonoids.