Diagnosis of osteomyelitis presents a formidable challenge. Lack of pathognomonic clinical sign (s) and diagnostic tests that can diagnose osteomyelitis at an early stage contribute to this difficulty. If the diagnosis is not made early, the disease becomes very difficult to eradicate and can lead to limb threatening and potentially life-threatening complications. Staphylococcus aureus is the most common organism causing osteomyelitis. Raman Spectroscopy provides information about molecular vibration that could potentially be considered as a spectral signature for cellular changes in specific pathologic conditions. In this study we describe a technique using Raman spectroscopy that could potentially be used to diagnose staphylococcal osteomyelitis. Human bone samples were co-cultured with Staphylococcus aureus (S. aureus) and the effects of bacterial growth on bone quality were monitored using Raman spectroscopy. A major drop in the bone mineral quality and crystallinity was observed in the infected bones compared to the controls. S. infection was also found to be cross-linking to the collagen. Our study shows that specific spectral signatures are present for the cause as well as the effect of staphylococcal osteomyelitis, opening the possibility of developing a useful diagnostic modality for early and rapid diagnosis of this condition.