Tumor growth is regulated by a diverse set of extracellular influences, including paracrine crosstalk with stromal partners, and biophysical interactions with surrounding cells and tissues. Studies elucidating the role of physical force and the mechanical properties of the extracellular matrix (ECM) itself as regulators of tumor growth and invasion have been greatly catalyzed by the use of in vitro three-dimensional (3D) tumor models. These systems provide the ability to systematically isolate, manipulate, and evaluate impact of stromal components and extracellular mechanics in a platform that is both conducive to imaging and biologically relevant. However, recognizing that mechanoregulatory crosstalk is bi-directional and fully utilizing these models requires complementary methods for in situ measurements of the local mechanical environment. Here, in 3D tumor/fibroblast co-culture models of pancreatic cancer, a disease characterized by its prominent stromal involvement, we evaluate the use of particle-tracking microrheology to probe dynamic mechanical changes. Using videos of fluorescently labeled polystyrene microspheres embedded in collagen I ECM, we measure spatiotemporal changes in the Brownian motion of probes to report local ECM shear modulus and microheterogeneity. This approach reveals stiffening of collagen in fibroblast co-cultures relative to cultures with cancer cells only, which exhibit degraded ECM with heterogeneous microstructure. We further show that these effects are dependent on culture geometry with contrasting behavior for embedded and overlay cultures. In addition to potential application to screening stroma-targeted therapeutics, this work also provides insight into how the composition and plating geometry impact the mechanical properties of 3D cell cultures that are increasingly widely used in cancer biology.