Osteomyelitis, a severe bone infection, poses a significant therapeutic challenge in both human and veterinary medicine, especially due to the increasing prevalence of antibiotic-resistant pathogens like methicillin-resistant Staphylococcus aureus (MRSA). Conventional treatments, including surgical debridement and systemic antibiotics, often prove inadequate due to the ability of bacteria to form biofilms and evade host immune responses. Antimicrobial peptides (AMPs), such as LL-37 and β-defensins, have emerged as a promising alternative therapeutic strategy. AMPs exhibit broad-spectrum antimicrobial activity, including efficacy against resistant strains, and possess immunomodulatory properties that can promote bone regeneration. This article comprehensively reviews AMP applications in treating osteomyelitis across both human and veterinary medicine. We discuss diverse therapeutic approaches, including free AMPs, their conjugation with biomaterials such as collagen and chitosan to enhance delivery and stability, and the development of AMP-based nanoparticles. Furthermore, we analyze preclinical and clinical findings, highlighting the efficacy and safety of AMPs in combating osteomyelitis in both human and animal patients. Finally, we explore future perspectives and challenges, such as optimizing delivery, stability, and efficacy, while minimizing cytotoxicity, and in translating AMP-based therapies into clinical practice to effectively manage this debilitating disease.