Bone is a nanocomposite material where the hard inorganic (hydroxyapatite crystallites) and organic (collagen fibrils) components are hierarchically arranged in the nanometer scale. Bone quality is dependent on the spatial distributions in the shape, size and composition of bone constituents (mineral, collagen and water). Bone hardness is an important property of bone, which includes both elastic and plastic deformation. In this study, a microhardness test was performed on a deer bone samples. The deer tibia shaft (diaphysis) was divided into several cross-sections of equal thickness; samples were prepared in untreated, boiled water treatment (100 °C for 30 min) and sodium hypochlorite (NaOCl) treatment conditions. Microhardness tests were performed on various regions of the tibial diaphysis to study the heterogeneous characteristics of bone microhardness and highlight the role of the organic matrix in bone hardness. The results indicated that boiled water treatment has a strong negative correlation with bone hardness. The untreated bone was significantly (+20%) harder than the boiled-water-treated bone. In general, the hardness values near the periosteal surface was significantly (23 to 45%) higher than the ones near the endosteal surface. Samples treated with NaOCl showed a significant reduction in hardness.