Failures of dental composite restorative procedures are largely attributed to the degradation of dentin–resin interface components. Biomodification of dentin using bioactive agents may improve the quality and durability of the dentin–resin bonds. The aim of this study was to nanomechanically assess the reduced modulus of elasticity (Er) and nano-hardness (H) of major components of the dentin–resin interface (hybrid layer, adhesive layer and underlying dentin) biomodified by collagen cross-linkers at 24h, 3 and 6 months following restorative procedure. Demineralized dentin surfaces were biomodified with 5% glutaraldehyde (GD) or 6.5% grape seed extract (GSE) prior to placement of adhesive systems and composite resin. Nano-measurements of the interface components in a fluid cell showed that both agents increased the Er and H of underlying dentin after 3 and 6 months when compared to a control. The mechanical properties of the adhesive and hybrid layers decreased over time. Biomodification of the dentin–resin interface structures using GD and GSE can increase the mechanical properties of the interface over time and may contribute to the long-term quality of adhesive restorations.