In this study, Eucalyptus nitens species was nano-characterized to determine variability in nanomechanical properties within the cellular ultra-structure between the bark and wood fibers. Three factors, including site (2 levels), family (2 levels) and fiber type (bark and wood) were analyzed using three response variables, including the elastic modulus (E), hardness (H) and ductility ratio (E/H) in the middle lamella (ML) and the cell wall within the S2 layer. The results indicated significant differences for ES2 and H-S2 when comparing fiber types: E-S2 approximate to 12.52 GPa and H-S2 approximate to 0.31 GPa for wood fiber and E-S2 approximate to 10.81 GPa and H-S2 approximate to 0.26 GPa for bark fiber. There is not statistically significant difference in ductility ratio (E/H) in S2 and ML between fiber types. These results indicate that bark and wood fibers can be used together or separately in the development of new composite materials and engineering products.