The tissue level mechanical properties of bone are a product of bone quantity and quality. Although bone quantity is easily defined, bone quality is much harder to define as it includes contributions from both bone chemistry and structure. This study investigated the chemical and nanostructural properties of rat cortical bone in order to determine how they are altered by osteoporosis and subsequent pharmaceutical treatments, including sodium fluoride, alendronate and parathyroid hormone. As part of this study, reflection-based Fourier Transform Infrared Microspectroscopy was developed to enable spatially resolved imaging of the chemical properties of bone specimens in the form of polished blocks. In addition, simultaneous microbeam Small and Wide Angle X-ray Scattering mapping was developed to investigate the collagen orientation, degree of orientation, D-spacing, and the apatite crystal geometry in bone specimens. Results from cortices of rat femurs showed that tissue mineralization and mineral crystallinity differed between intracortical and circumferential bone on both endosteal and periosteal surfaces, but no differences in nanostructural properties between these two types of bone were found. In osteoporotic bone, greater differences in mineralization and carbonate substitution were observed between intracortical and periosteal bone. Additionally, the collagen fibrils in intracortical bone were found to have an unusually high alignment and changes were found in the geometry of the mineral crystals of newly formed periosteal bone. Upon treatment with sodium fluoride, newly formed bone had greater carbonate substitution and crystallinity of apatite compared to osteoporotic bone, suggesting that sodium fluoride affects the rate of mineral maturation in newly formed bone. Changes to the collagen alignment induced by osteoporosis were mitigated by sodium fluoride and alendronate, but not by parathyroid hormone. However, both alendronate and parathyroid hormone mitigated alterations to the crystal geometry. Together, these results show that osteoporosis and pharmaceutical treatments affect the chemical composition and nanostructural properties of rat cortical bone. A further understanding between the interplay of the parameters that define bone quality, the ultimate tissue level mechanical properties, and fracture resistance of bone may lead to more potent pharmaceutical drugs to recuperate compromised bone quality.