The objective of this study was to investigate the osteogenic and antimicrobial effect of bioactive glass S53P4 incorporated into β-tricalcium phosphate (β-TCP) scaffolds in vitro and the bone neoformation in vivo. β-TCP and β-TCP/S53P4 scaffolds were prepared by the gel casting method. Samples were morphologically and physically characterized through X-ray diffraction (XRD) and scanning electron microscope (SEM). In vitro tests were performed using MG63 cells. American Type Culture Collection reference strains were used to determine the scaffold’s antimicrobial potential. Defects were created in the tibia of New Zealand rabbits and filled with experimental scaffolds. The incorporation of S53P4 bioglass promotes significant changes in the crystalline phases formed and in the morphology of the surface of the scaffolds. The β-TCP/S53P4 scaffolds did not demonstrate an in vitro cytotoxic effect, presented similar alkaline phosphatase activity, and induced a significantly higher protein amount when compared to β-TCP. The expression of Itg β1 in the β-TCP scaffold was higher than in the β-TCP/S53P4, and there was higher expression of Col-1 in the β-TCP/S53P4 group. Higher bone formation and antimicrobial activity were observed in the β-TCP/S53P4 group. The results confirm the osteogenic capacity of β-TCP ceramics and suggest that, after bioactive glass S53P4 incorporation, it can prevent microbial infections, demonstrating to be an excellent biomaterial for application in bone tissue engineering.