Novel biomaterials provide fundamental platforms for diagnosing diseases at the cellular level, and also present opportunity to deliver therapy to unhealthy cells without damaging healthy cells. Among numerous biomaterials, magnetically-aligned collagen matrices offer advantages in fundamental biological studies, such as imaging and cell therapy, such as hyperthermia, magneto-mechanical rupture, and drug delivery. Understanding and predicting structure, as well as mechanical and magnetic properties, of these magnetically-aligned matrices all play a crucial role in successfully producing and implementing biological treatment techniques. Experimentally, highly uniform magnetic nanowires (MNWs) were fabricated using template assisted electrochemical deposition technique. After coating MNWs with NH2-PEG-COOH, they were incubated in collagen type I with/without CDI crosslinker at different MNWs concentrations. By applying a weak magnetic field during gelation, the collagen matrices were aligned, providing a bi-directional alignment of the collagen fibrils without any extra preparation processes. Confocal reflectance microscopy and differential interference contrast (DIC) microscopy were used to visualize the structure of matrices. Atomic force microscopy (AFM) was used to image the structure and correlate this structure to the mechanical properties of each matrix measured using AFM contact resonance viscoelastic mapping and AFM nanoindentation techniques. AFM nanomechanical measurements show enhancement in mechanical properties of magnetically-aligned collagen matrices when the CDI crosslinker was used. For magnetic properties, vibrating sample magnetometry measurements were analyzed using First Order Reversal Curves (FORC), such that the magnetic properties of the matrices were quantitatively and qualitatively investigated, confirming that the strongest matrices contained uni-directional alignment of MNWs in bi-directionally aligned collagen.