Quercetin 3-acetyl-7,3',4'-trisulphate (ATS) and quercetin 3,7,3',4'-tetrasulphate (QTS) obtained from Flaveria bidentis (Asteraceae) were investigated in vitro for anticoagulant activity. Three different concentrations of each flavonoid were assayed at different incubation times, showing at 1 mM significant prolongation on the activated partial thromboplastin time (APTT), less on the prothrombin time (PT), and no effect on the thrombin time (TT). In order to define the action mechanism of the anticoagulant activity, all coagulation factors were evaluated and no important activity decrease was observed, indicating that another mechanism is involved. Thus, thrombin inhibition mediated by antithrombin III (ATIII) and heparin cofactor II (HCII) activation was investigated in comparison to the physiological activators, heparin and dermatan sulphate (DS), respectively. As a conclusion, no activation on ATIII for neither flavonoids was observed. On the contrary, QTS much more than ATS produced an activation on HCII comparable to the one of DS, indicating that these flavonoids act as agonists of this inhibitor. A plausible explanation of the effects of both flavonoids could be due to the different degree of sulphation of these molecules. According to the results obtained, and taking in account the high solubility of these natural products in aqueous media and the nontoxic nature of this family of compounds, further investigation on the antithrombotic effects of these flavonoids are merited.