Bone infections, particularly osteomyelitis, are complex and difficult-to-treat conditions that significantly impact orthopedic outcomes. Staphylococcus aureus remains the primary pathogen associated with chronic osteomyelitis, exploiting biofilm formation to persist in bone tissue despite immune defenses and antibiotic interventions. Biofilms are structured microbial communities encased in an extracellular matrix that can form on both device surfaces and native bones, enabling bacteria to evade host immune responses and resist antimicrobial therapies. Current research has revealed biofilm reservoirs within the bone, including staphylococcal abscesses in soft tissue, biofilm layers on necrotic tissue, and colonization of the osteocyte lacuno-canalicular network (OLCN) within the cortical bone. Despite significant advancements in the understanding of the biofilm-driven pathogenesis of osteomyelitis, clinical management remains challenging. Traditional distinctions between acute and chronic osteomyelitis are being reconsidered, with an increasing emphasis on immunoproteomic profiling to guide diagnosis and treatment. Novel approaches to local antibiotic therapy, such as antimicrobial-coated implants and biofilm-disrupting agents, offer promising avenues for enhancing treatment efficacy. This chapter explores the mechanisms of biofilm-mediated bone infections, immune response dynamics, and the latest therapeutic strategies for managing biofilm-associated osteomyelitis, aiming to provide more effective and durable solutions for these debilitating infections.