The effect of varying electrospinning parameters is reported for the production of collagen nanofibers from acetic acid with controlled fiber diameter, orientation, and mechanical properties. Nanofibers with a range of diameters of 175–400 nm are obtained by varying either the voltage or the flow rate. An increase in nanofiber alignment is observed by increasing injection flow rate. Mechanical testing of these fibers reveals that the elasticity modulus can be tuned in the range of 2.7–4.1 MPa by the selection of the crosslinking method. Fourier transform infrared spectroscopy reveals that the secondary structure of collagen is preserved after electrospinning and crosslinking. Lastly, in vitro testing reveals that a high number of fibroblasts attach to the collagen matrices indicating, that they are suitable for mammalian cell culture.