To evaluate the effectiveness of locally applied vancomycin powder at different times postinfection in a contaminated traumatic animal model. This study used an established segmental defect rat femur model contaminated with Staphylococcus aureus UAMS-1 followed by treatment at 6 or 24 hours postinfection. Three treatments were evaluated: debridement and irrigation alone (control group) or in combination with either vancomycin powder or vancomycin-impregnated poly(methyl methacrylate) beads. Serum vancomycin levels were determined at scheduled time points over 14 days; bone, surrounding muscle, and implants were harvested for bacterial and inflammatory analyses. Locally applied vancomycin powder and impregnated beads significantly reduced bacteria both within the bone and implant when treatment was performed at 6 hours. Delaying treatment to 24 hours significantly reduced the therapeutic efficacy of locally applied vancomycin of both groups. Serum vancomycin levels were detectable in all animals treated with vancomycin powder at 24 hours, but absorption was negligible from beads. At 14 days, vancomycin was detectable in the surrounding musculature of all animals and in serum of 20% of animals treated with vancomycin powder. This study suggests that vancomycin powder is a promising adjunctive therapy for preventing infection in traumatic wounds when treatment is performed early. This time-dependent effectiveness of vancomycin powder is similar to that observed with systemic and other local delivery adjuncts, which is likely attributable to biofilm formation after contamination, conferring intrinsic recalcitrance to antimicrobials.