Abstract Quantitative contrast-enhanced ultrasound plays an important role in tumor characterization and treatment assessment. Besides established functional ultrasound techniques, ultrasound molecular imaging using microbubbles targeted to disease-associated markers is increasingly being applied in pre-clinical studies. Often, repeated injections of non-targeted or targeted microbubbles during the same imaging session are administered. However, the influence of repeated injections on the accuracy of the quantitative data is unclear. Therefore, in tumor-bearing mice, we investigated the influence of multiple injections of non-targeted microbubbles (SonoVue) on time to peak and peak enhancement in liver and tumor tissue and of vascular endothelial growth factor receptor 2 (VEGFR2)-targeted contrast agents (MicroMarker) on specific tumor accumulation. We found significantly decreasing values for time to peak and a tendency for increased values for peak enhancement after multiple injections. Repeated injections of VEGFR2-targeted microbubbles led to significantly increased tumor accumulation, which may result from the exposure of additional binding sites at endothelial surfaces caused by mechanical forces from destroyed microbubbles.