Staphylococcus aureus, the principal causative agent of osteomyelitis, can be internalized by osteoblasts and thereby escape from immune phagocytes and many kinds of antibiotics. To deliver antibiotics into osteoblasts to kill S. aureus in the intracellular environment, we developed gentamicin-loaded chitosan nanoparticles and evaluated their intracellular bactericidal effect. We found decreased numbers of S. aureus cells in infected osteoblasts treated with gentamicin-loaded chitosan nanoparticles. The cytotoxicity of the nanoparticles was evaluated by CCK-8 assay. There was no significant viability decrease at all tested concentrations. In conclusion, our results provide evidence for the potential use of gentamicin-loaded chitosan nanoparticles to enhance the delivery of gentamicin into cells and for their antibacterial effect against internalized S. aureus in the intracellular environment of osteoblasts.