Endothelial defects (ED) and the usage of interposition vein grafts (IVG) are known risk factors for free flap failure. This experimental study aimed to compare both situations of thrombus formation and fluorescence angiographic behavior. Indocyanine green videoangiography (ICGVA) with the FLOW 800 tool was systematically performed in groups I = ED, II = IVG, and III = ED and IVG (each n = 11). ICGVA was able to detect thrombosis in five animals and safely ruled it out in 26 with two false-positive cases (sensitivity, specificity, and positive and negative predictive values were 100%, 90%, 62%, and 100%, respectively). The difference between visually and ICGVA-assisted ED measurements was significant (p = 0.04). The areas of thrombosis showed no significant difference. Moreover, ICGVA detected a decrease of all parameters at the ED area and/or within the IVG section in all groups. The presence of an endothelial defect had a higher impact on thrombus formation than the IVG usage. ICGVA is qualitatively able to detect endothelial defects and clinically evident thrombosis. However, the quantitative values are not yet attributable to one of the clinical scenarios that may jeopardize free flap transfer.