Microbubbles (MB) have previously demonstrated their great potential to enhance sonothrombolysis (STL) in vascular occlusions. Nonetheless the impact of MB-mediated STL on the fibrin network of clots has not been investigated up to now. The aim of this study was to evaluate the ability of combined ultrasound (US) and MB to degrade the fibrin in the presence or absence of a thrombolytic drug. Human blood clots containing radiolabeled-fibrin were exposed to different combinations of recombinant tissue plasminogen activator (rtPA), US (1.0 MHz; 1.3 MPa; 0.08% duty cycle) and MB. Three different techniques were used for the assessment of clot lysis: diameter loss, release of radioactive-fibrin degradation products (FDP) and D-Dimer assay. On one hand, the combination US þ MB without rtPA induced a diameter loss (0.18 ± 0.01 mm), but no fibrin degradation, as revealed by the absence of radioactive-FDP release (0.5 ± 0.1%). On the other hand, the combination rtPA þ US þ MB enhanced both the diameter loss (0.77 ± 0.07 mm) and fibrin degradation (50.3 ± 2.4%) compared to rtPA alone (0.24 ± 0.01 mm and 36.5 ± 1.7%; p < 0.001), thus demonstrating a strong synergistic effect. The D-Dimer assay further confirmed the radioactivity results. Sonothrombolysis with MB greatly improves rtPA-mediated clot lysis and fibrin degradation was shown to be a key factor of the process. Studies addressing the clinical impact of this technique are warranted.