The delivery of nanoparticles to tumors has been shown preclinically to be improved by microbubble-mediated ultrasound. However, the mechanisms and biological effects are not fully understood. In this study, we explored the influence of the tumor microenvironment on nanoparticle uptake and microdistribution both with and without ultrasound and microbubble treatment. Three murine tumor models, KPC (pancreatic ductal adenocarcinoma), 4T1 (triple negative mammary carcinoma) and CT26 (colon carcinoma), were characterized with respect to extracellular matrix composition, tumor stiffness and perfusion. KPC and 4T1 tumors presented higher levels of collagen and hyaluronic acid and were stiffer compared to CT26, whereas all three tumors had similar levels of sulfated glycosaminoglycans. Furthermore, the 4T1 tumors appeared poorly vascularized with a lower cell density compared to KPC and CT26. All three tumors presented similar nanoparticle uptake, but extravasated nanoparticles traveled significantly shorter in KPC tumors compared to 4T1 and CT26. The effect of ultrasound and microbubble treatment on the tumor uptake and penetration of polymer nanoparticles into the extracellular matrix were evaluated using a treatment protocol previously shown to increase nanoparticle delivery to tumors. Interestingly, we found a significant increase in nanoparticle uptake in the soft CT26 tumor, but no effect of the ultrasound treatment in the stiff KPC and 4T1 tumors, suggesting that tumor stiffness is an important parameter for treatment with ultrasound and microbubbles. Ultrasound treatment resulted in a modest but not statistically significant improvement in nanoparticle penetration through the extracellular matrix. In tumors demonstrating increased uptake of nanoparticles following ultrasound treatment, the uptake correlated positively with blood volume. These findings emphasize the importance of taking the tumor microenvironment into consideration when optimizing ultrasound parameters for delivery of nanomedicine.