Background: When bacteria colony persist within a biofilm, suitable drugs are not yet available for the eradication of biofilm-producing bacteria. The aim of this study is to study the effect of magnetic nano-particles-induced hyperthermia on destroying biofilm and promoting bactericidal effects of antibiotics in the treatment of osteomyelitis. Methods: Sixty 12-weeks-old male Wistar rats were used. A metallic 18G needle was implanted into the bone marrow cavity of distal femur after the injection of Methicillin-sensitive Staphylococcus aureus (MSSA). All animals were divided into 5 different treatment modalities. The microbiological evaluation, scanning electron microscope examination, radiographic examination and then micro-CT evaluation of peri-implant bone resorption were analyzed. Results: The pathomorphological characteristics of biofilm formation were completed after 40-days induction of osteomyelitis. The inserted implants can be heated upto 75 °C by magnetic heating without any significant thermal damage on the surrounding tissue. We also demonstrated that systemic administration of vancomycin [VC (i.m.)] could not eradicate the bacteria; but, local administration of vancomycin into the femoral canal and the presence of magnetic nanoparticles hyperthermia did enhance the eradication of bacteria in a biofilm-based colony. In these two groups, the percent bone volume (BV/TV: %) was significantly higher than that of the positive control. Conclusions: For the treatment of chronic osteomyelitis, we developed a new modality to improve antibiotic efficacy; the protection effect of biofilms on bacteria could be destroyed by magnetic nanoparticles-induced hyperthermia and therapeutic effect of systemic antibiotics could be enhanced.