Aim: This study assessed the effect of the dentin depth and the application mode on the hardness and elastic modulus of the adhesive layer. Materials and Methods: Occlusal surfaces of 48 caries-free human third molars were removed, at two levels: Superficial and deep dentin. For each type of surface, the test specimens were randomly divided into groups which underwent the application: A conventional two-step adhesive system (Adper™ Single Bond [SB]) and self-etch adhesives system (Adper™ SE Plus [SE] and AdheSE® [AD]). The adhesives applied were active or passive. Composite build-ups were constructed incrementally. The teeth were sectioned, embedded, and polished. The nanoindentation test was performed in the adhesive layer. The results were analyzed by ANOVA and Tukey tests. Results: In the adhesive layer, the higher hardness (0.307 ± 0.006 GPa) and elastic modulus (4.796 ± 0.165 GPa) of SE were obtained in superficial dentin in passive application. The elastic modulus of SE (4.115 ± 0.098 GPa) was lowest in active application in superficial dentin. The active application significantly increased the hardness of the SB in the deep dentin (0.011 ± 0.314 GPa) compared the superficial dentin (0.280 ± 0.010 GPa). For the AD, only the mode of application was statistically significant (P=0.0041) for the hardness, active application (0.289 ± 0.015 GPa) being higher than passive application (0.261 ± 0.013 GPa) (P=0.0042) in deep dentin. Conclusion: The experimental results reveal that the mechanical properties were influenced for the application mode of adhesive systems and dentin depth.