Bone and intramedullary bacterial infections are one of the most serious complications of the surgical repair of fractures. To reduce the incidence of implant-related infections, several biomaterial surface treatments with integrated antibiotics, antiseptics, or metal ions have been developed for implants. In this study, we evaluated the antibacterial activity and biocompatibility of 317L stainless steel containing 4.5% copper alloy (317L-Cu) in vitro and in vivo using an animal model. Common pathogens of implant-related infections are Staphylococcus aureus and Escherichia coli, which were injected into implant materials to study their antimicrobial potential. We compared antimicrobial potential of 317L-Cu with 317L stainless steel (317L) and titanium (Ti-6Al-4V) alloys as controls. Compared with controls, 317L-Cu materials inhibited colonization by both bacteria in vitro and in vivo. Compared with 317L and Ti-6Al-4V controls, 317L-Cu showed no significant difference in colony formation of osteoblast-like cells on metal surfaces after 72 h of incubation in vitro. Metal screws containing these materials were also made for our vivo study in a rabbit model. Tissue-implants were analyzed for infection and inflammatory changes by hematoxylin-eosin staining of implants in bone. The screw tract inflammation and infection of 317L-Cu was minimal, although some inflammatory cells gathered at acutely infected sites. In addition, after materials had been implanted for 14 days in vivo, the expression of insulin-like growth factor-1 (IGF-1) in osteoblasts around 317L-Cu screws tracts had increased compared with 317L and Ti-6Al-4V controls. Overall, 317L-Cu demonstrated strong antimicrobial activity and biocompatibility in vitro and in vivo and may be used as a biomaterial to reduce implant-related infections.