Museum anthropology collections continue to provide insight into human experiences and disease in the past, especially in the era of biomolecular research. Molecular investigations of museum collections have increased dramatically since the recent high throughput sequencing revolution. However, basic biological questions regarding the preservation of museum biomolecules and the kind of information biomolecular substrates can provide need to be clarified. In this dissertation, four studies address the challenges to conducting molecular research with museum collections, in particular, the impact of unique acquisition and treatment histories of collections, and how museum biomolecules can be used to answer biological questions. Collections from the Smithsonian Institution’s National Museum of Natural History (NMNH) Anthropology department are utilized in this dissertation to investigate: specific mechanisms and approaches to curating museum biomolecules (Chapter Two); the recovery of DNA among globally representative skeletal populations that have specific and unique acquisition and treatment histories (Chapter Three); the diagnostic potential of dental calculus to confirm COD of either syphilis or tuberculosis, using the Terry Collection (Chapter Four); and, finally, the end of life circumstance of a single individual using historic and collection contextualization, skeletal assessment, and oral microbiome molecular methods (Chapter Five).