Collagen thin films consisting of randomly oriented and oriented collagen fibrils/fibers are fabricated by hydrodynamic flow and spin coating, and then they are characterized by atomic force microscopy (AFM). Fibroblasts are cultured on these films in order to correlate their morphology and alignment, which are assessed with fluorescence and AFM imaging with different film characteristics. The results showed that the formed films could be used as substrates for culturing cells. Furthermore, cells reacted to film nanocharacteristics and especially to the orientation of fibrils/fibers. The investigation of the influence that the substrate nanotopography has on cells will help to elucidate the mechanisms of cell–biomaterial interactions, and will enable the design of intelligent coatings for implants and tissue engineering purposes.