Our previous study (Z. X. Peng et al., Carbohydr. Polym. 81:275-283, 2010) demonstrated that water-soluble quaternary ammonium salts, which are produced by the reaction of chitosan with glycidyl trimethylammonium chloride, provide chitosan derivatives with enhanced antibacterial ability. Because biofilm formation is believed to comprise the key step in the development of orthopedic implant-related infections, we further evaluated the efficacy of hydroxypropyltrimethyl ammonium chloride chitosan (HACC) with different degrees of substitution (DS; referred to as HACC 6%, 18%, and 44%) in preventing biofilm formation on a titanium surface. We used a tissue culture plate method to quantify the biomass of Staphylococcus epidermidis and Staphylococcus aureus biofilms and found that HACC, especially HACC 18% and 44%, significantly inhibited biofilm formation compared to the untreated control, even at concentrations far below their MICs (P < 0.05). Scanning electron microscopy showed that inhibition of biofilm formation on titanium increased dramatically with increased DS and HACC concentrations. Confocal laser scanning microscopy indicated that growth of a preexisting biofilm on titanium was inhibited by concentrations of HACC 18% and 44% below their minimum biofilm eradication concentrations. We also demonstrated that HACC inhibited the expression of icaA, which mediates the production of extracellular polysaccharides, both in new biofilms and in preexisting biofilms on titanium. Our results indicate that HACC may serve as a new antibacterial agent to inhibit biofilm formation and prevent orthopedic implant-related infections.