Infection of open tibial fractures with contamination remains a challenge for orthopedic surgeons. Local use of antibiotic-impregnated polymethylmethacrylate (PMMA) beads and blocks is a widely used procedure to reduce the risk of infection. However, the development of antibiotic-resistant organisms make the management of infection more difficult. Our in vitro study demonstrated that quaternized chitosan (hydroxypropyltrimethyl ammonium chloride chitosan [HACC])-loaded PMMA bone cement exhibits strong antibacterial activity toward antibiotic-resistant bacteria. Therefore, the present study aimed to investigate the in vivo antibacterial activity of quaternized chitosan-loaded PMMA. Twenty-four adult female New Zealand White rabbits were used in this study. The right proximal tibial metaphyseal cavity was prepared, 10(7) CFU of methicillin-resistant Staphylococcus epidermidis was inoculated, and PMMA-only, gentamicin-loaded PMMA (PMMA-G), chitosan-loaded PMMA (PMMA-C), or HACC-loaded PMMA (PMMA-H) bone cement cylinders were inserted. During the follow-up period, the infections were evaluated using X rays on days 21 and 42 and histopathological and microbiological analyses on day 42 after surgery. Radiographic indications of bone infections, including bone lysis, periosteal reactions, cyst formation, and sequestral bone formation, were evident in the PMMA, PMMA-G, and PMMA-C groups but not in the PMMA-H group. The radiographic scores and gross bone pathological and histopathological scores were significantly lower in the PMMA-H group than in the PMMA, PMMA-G, and PMMA-C groups (P < 0.05). Explant cultures also indicated significantly less bacterial growth in the PMMA-H group than in the PMMA, PMMA-G, and PMMA-C groups (P < 0.01). We concluded that PMMA-H bone cement can inhibit the development of bone infections in this animal model inoculated with antibiotic-resistant bacteria, thereby demonstrating its potential application for treatment of local infections in open fractures.