Interfacial dynamics, assembly processes, and changes in nanostructures and mechanical properties of Bombyx mori silk fibroin (SF) proteins under varying degrees of nanoconfinement without and with lateral shear are investigated. When only compressive confinement forces were applied, SF proteins adsorbed on the surfaces experienced conformational changes following the Alexander-de Gennes theory of polymer brushes. By contrast, when SF proteins were exposed to a simultaneous nanoconfinement and shear, remarkable changes in interaction forces were observed, displaying the second order phase transitions, which are attributed to the formation of SF micelles and globular superstructural aggregates via hierarchical assembly processes. The resultant nanostructured SF aggregates show several folds greater elastic moduli than those of SF films prepared by drop-casting and compression-only and even degummed SF fibers. Such a striking improvement in mechanical strength is ascribed to a directional organization of β-sheet nanocrystals, effectively driven by nanoconfinement and shear stress-induced stiffing and ordering mechanisms.