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Purified collagen possesses weak mechanical properties, hindering its broad application in tissue engineering. Strategies based on manipulating the hydrogel to induce fiber formation or incorporate nanomaterials have been proposed to overcome this issue. Herein, we use a microfluidic device to fabricate, for the first time, collagen hydrogels with aligned and oriented fibers doped with gold nanoparticles and carbon nanotubes. Results based on rheology, atomic force microscopy, and scanning electron microscopy reveal the formation of aligned and oriented collagen fibers possessing greater rigidity and stiffness on the doped hydrogels in comparison with native collagen. The mechanical properties of the hydrogels increased with the nanomaterial loading percentage and the stiffest formulations were those prepared in the presence of carbon nanotubes. We further evaluate the in vitro response of NIH-3T3 fibroblasts to the change in stiffness. The cells were found to be viable on all substrates with directional cell growth observed for the carbon nanotube-doped collagen fibers. No significant differences in the cell area, aspect ratio, and intensification of focal adhesions driven by the increase in stiffness were noted. Nonetheless, fibroblast proliferation and secretion of TGF-β1 were greater on the hydrogels doped with carbon nanotubes. This nanomaterial-collagen composite provides unique features for cell and tissue substrate applications.