Osteomyelitis is one of the most common inflammatory bone diseases caused by gram-positive bacteria. Although cefazolin is widely used as an osteomyelitis antibiotic against Staphylococcus aureus, its thermolabile properties may decrease its antibiotic activity when incorporated into bone cement, such as polymethyl methacrylate. Thus, to fully characterize the loss of antibiotic activity during the fused deposition modeling 3D-printing processes, we systematically evaluated the antibacterial activity of a cefazolin-loaded polycaprolactone (PCL)-based scaffold at varying 3D printing temperatures against S. aureus. In vitro antibacterial activity analysis revealed that cefazolin-loaded PCL scaffolds printed between 60 °C and 120 °C could maintain the antibacterial activity, whereas the antibacterial activity of scaffolds printed between 140 °C and 160 °C was significantly decreased. In addition, the therapeutic potential of the 3D-printed, scaffolds was assessed based on serum interleukin-6 (IL-6) levels, micro-computed tomography (micro-CT), and histological evaluation using a rabbit model of S. aureus-induced chronic osteomyelitis. Our in vivo results demonstrated that the serum level of IL-6 decreased in the cefazolin-loaded PCL scaffold-transplanted group. Furthermore, micro-CT and histological analyses confirmed the decreased deformation and overgrowth of the bone. The utilization of a 3D-printable, cefazolin-loaded PCL scaffold offers a promising and distinct approach for the treatment of osteomyelitis.