Infection is an important medical problem associated with the use of bone allografts. To retard bacterial colonization, we have recently reported on the modification of bone allografts with the antibiotic vancomycin (VAN). In this report, we examine the ability of this antibiotic-modified allograft to resist bacterial colonization and biofilm formation. When antibiotic was coupled to the allograft, a uniform distribution of the antibiotic was apparent. Following challenges with Staphylococcus aureus for 6 h, the covalently bonded VAN decreased colonization as a function of inoculum, ranging from 0.8 to 2.0 log(10) CFU. Furthermore, the VAN-modified surface resisted biofilm formation, even in topographical niches that provide a protected environment for bacterial adhesion. Attachment of the antibiotic to the allograft surface was robust, and the bonded VAN was stable whether incubated in aqueous media or in air, maintaining levels of 75 to 100% of initial levels over 60 days. While the VAN-modified allograft inhibited the Gram-positive S. aureus colonization, in keeping with VAN's spectrum of activity, the VAN-modified allograft was readily colonized by the Gram-negative Escherichia coli. Finally, initial toxicity measures indicated that the VAN-modified allograft did not influence osteoblast colonization or viability. Since the covalently tethered antibiotic is stable, is active, retains its specificity, and does not exhibit toxicity, it is concluded that this modified allograft holds great promise for decreasing bone graft-associated infections.