The purpose of our study was to investigate the antibacterial effect of a spacer (Ti6Al4V) coated with 4x Cu-TiO2 in an animal model simulating an acute periprosthetic infection by Staphylococcus aureus. Ti6Al4 bolts contaminated with Staphylococcus aureus were implanted into the femoral condyle of rabbits (n = 36) divided into 3 groups. After one week in group 1 (control) the bolts were removed without any replacement. In group2 Ti6Al4V bolts with a 4x Cu-TiO2 coating and in group 3 beads of a gentamicin-PMMA chain were imbedded into the borehole. Microbiological investigation was performed at the primary surgery, at the revision surgery and after scarification of the rabbits 3 weeks after the first surgery. Blood tests were conducted weekly. The initial overall infection rate was 88.9%. In group 2 and 3 a significant decrease of the infection rate was shown in contrast to the control group. The C-reactive protein (CRP) levels declined one week after the first surgery except in the control group where the CRP level even increased. This is the first in vivo study that demonstrated the antibacterial effects of a fourfold Cu-TiO2 coating. For the future, the coating investigated could be a promising option in the treatment of implant-associated infections.