Bone mechanical properties at the tissue level are affected due to changes of bone components and structure at the nanoscale. Studying bone structure and material properties at various length scales is therefore important to attain a better understanding of the origin of bone mechanical properties and discern the origin changes with age and disease as well as their influence and impact on bone fracture. Cantilever-based nanoindentation Atomic Force Microscopy offers a possibility to interrogate bone mechanical properties at the nanoscale. It has previously been suggested that the heterogeneity of bone at the lamellar level may be a critical factor influencing bone fracture toughness. Within this thesis this suggestion was further investigated. Thirty bone samples from the human femur with existing fracture toughness measurements were obtained from a collaborator and were investigated using Atomic Force Microscopy in cantilever-based nanoindentation mode and Second Harmonic Generation Microscopy to interrogate collagen fibril orientation. The heterogeneity in indentation modulus between osteonal lamellae showed a potential statistically non-significant trend due to age. Similarly, collagen fibril orientation distributions did not change significantly with age. However, the Atomic Force Microscope imaging of osteonal lamellae revealed a distinctive lamellar structure (height topography) even when sampled were immersed in Phosphate-Buffered Saline for more than 24h in contrast to a previous study. Most importantly fracture initiation toughness and resistance to crack propagation correlate significantly with high degree of nanoelasticity heterogeneity between osteonal lamellae and interlamellar areas. However, fracture toughness parameters do not correlate at a significant level with the collagen fibril orientation distributions. This shows that indeed the modulation of stiffness at the lamellar level is influencing fracture toughness, but it does not seem to depend on age.