Although total joint replacement has become commonplace in recent years, bacterial infection remains a significant complication following this procedure. One approach to reduce the incidence of joint replacement infection is to add antimicrobial agents to the bone cement used to fix the implant. In this in vitro study, we investigated the use of chitosan nanoparticles (CS NP) and quaternary ammonium chitosan derivative nanoparticles (QCS NP) as bactericidal agents in poly(methyl methacrylate) (PMMA) bone cement with and without gentamicin. The antibacterial activity was tested against Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). A 10(3)-fold reduction in the number of viable bacterial cells upon contact with the surface was achievable using QCS NP at a nanoparticle/bone cement weight ratio of 15%. The inhibition of S. aureus and S. epidermidis growth on the surface of the CS NP and QCS NP-loaded bone cements was clearly shown using the LIVE/DEAD Baclight bacterial viability kits and fluorescence microscopy. The CS NP and QCS NP also provided a significant additional bactericidal effect to gentamicin-loaded bone cement. The antibacterial effectiveness remained high even after the modified bone cements had been immersed for 3 weeks in an aqueous medium. No cytotoxic effect of the CS NP- and QCS NP-loaded cements was shown in a mouse fibroblast MTT cytotoxicity assay. Mechanical tests indicated that the addition of the CS and QCS in nanoparticulate form allowed the retention of a significant degree of the bone cement's strength. These results indicate a new promising strategy for combating joint implant infection.