Microstructural anisotropy of tumor-associated matrix correlates with invasion of cancer cells into the surrounding matrix during metastasis. Here, we report the fabrication and characterization of a three-dimensional (3D) silk-fibroin/collagen-I bio-composite based cell-culture model that exhibits microstructural and biochemical anisotropy. Using RGD-deficient silk-fibroin fibers to confine collagen-I gelation, we develop a silk-fibroin/collagen-I (SFC) bio-composite in a one-step process allowing control over the microstructural and biochemical anisotropy and the pore-size. Two forms of the SFC bio-composite are reported: a sandwich (S_fc) configuration amenable to live-cell microscopy and an unsupported membrane (M_fc) for use as a scaffold. Both microscalar and macroscalar mechanical properties of the SFC bio-composite are characterized using atomic force microscope (AFM)-based indentation and tensile-testing. We find that the modulus of stiffness of both S_fc and M_fc can be controlled and falls in the physiological range of 5–20 kPa. Furthermore, the modulus of stiffness of M_fc exhibits a ~200% increase in axial direction of microstructure, as compared to lateral direction. This implies a highly anisotropic mechanical stiffness of the microenvironment. Live-cell morphology and migration studies show that both the morphology and the migration of NIH-3 T3 fibroblasts is anisotropic and correlates with microstructural anisotropy. Our results show that SFC bio-composite permits proliferation of cells in both S_fc and M_fc configuration, promotes cell-migration along the major axis of anisotropy and together with morphological and migration data, suggest a potential application of both the composite configurations as a biomimetic scaffold for tissue engineering applications.