Circulating thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1) are causal factors for thrombolytic failure. Therefore, we evaluated an antibody-engineered bispecific inhibitor against TAFI and PAI-1 (heterodimer diabody, Db-TCK26D6x33H1F7) in several mouse models of thrombosis and stroke. Prophylactic administration of the diabody (0.8mg/kg) in a thromboplastin-induced model of thromboembolism led to decreased lung fibrin deposition. In a model of cerebral ischemia/reperfusion, diabody administration (0.8mg/kg, 1h post occlusion) led to a mitigated cerebral injury with a 2.3-fold reduced lesion and improved functional outcomes. In a mouse model of thrombin-induced middle cerebral artery occlusion (MCAo), the efficacy of the diabody was compared to the standard thrombolytic treatment with recombinant tissue-type plasminogen activator (tPA). Early administration of diabody (0.8mg/kg, 20min post occlusion) caused a 2-fold decrease in brain lesion size, whereas that of tPA (10mg/kg) had a much smaller effect. Delayed administration of diabody or tPA (90min post occlusion) had no effect on lesion size, whereas the combined administration of diabody with tPA caused a 1.7-fold decrease in lesion size. In contrast to tPA, the diabody did not increase accumulative bleeding. In conclusion, administration of a bispecific inhibitor against TAFI and PAI-1 results in a prominent profibrinolytic effect in mice without increased bleeding.