We show that mechanical stiffness is a useful metric for characterizing complex collagen assemblies, providing insight about aggregation products and pathways in collagen-based materials. This study focuses on mechanically robust collagenous membranes produced by an electrochemical synthesis process. Changing the duration of the applied electric field, or adjusting the electrolyte composition (by adding Ca(2+), K(+), or Na(+) or by changing pH), produces membranes with a range of Young's moduli as determined from force-displacement measurements with an atomic force microscope. The structural organization, characterized by UV-visible spectroscopy, Raman spectroscopy, optical microscopy, and atomic force microscopy, correlates with the mechanical stiffness. These data provide insights into the relative importance of different aggregation pathways enabled by our multiparameter electrochemically induced collagen assembly process.