Periprosthetic joint infection (PJI) is a leading cause and major complication of joint replacement failure. As opposed to standard-of-care systemic antibiotic prophylaxis for PJI, we developed and tested titanium femoral intramedullary implants with titania nanotubes (TNTs) coated with the antibiotic gentamicin and slow-release agent chitosan through electrophoretic deposition (EPD) in a mouse model of PJI. We hypothesized that these implants would enable local gentamicin delivery to the implant surface and surgical site, effectively preventing bacterial colonization. In the mouse PJI model, C57BL/6 mice received implants with TNTs coated with chitosan (chitosan group; control group) or with TNTs coated with chitosan and gentamicin (chitosan + gentamicin group; experimental group). Following implant placement, the surgical site was inoculated with 1 × 10 CFUs of Xen36 bioluminescent Staphylococcus aureus. All the mice in the chitosan group and none in the chitosan + gentamicin group had evidence of infection based on CFU analysis and bioluminescence imaging through the 14-day assessment postsurgery. Correspondingly, scanning electron microscopy analysis at the implant surface demonstrated bacterial biofilm only in the chitosan group. Furthermore, periosteal reaction and peri-implant bone loss at the femur were significantly reduced in the chitosan + gentamicin group. The chitosan + gentamicin group had reduced pain behavior, improved weight-bearing, and increased weight compared to the chitosan-control group. This study provides preclinical evidence supporting the efficacy of implants with TNTs coated with chitosan and gentamicin through EPD for preventing bacterial colonization and biofilm formation in a mouse model of PJI.