Injectable hydrogels can serve as therapeutic vehicles and implants for the treatment of various diseases as well as for tissue repair/regeneration. In particular, the horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂)-catalyzed hydrogelation system has attracted much attention, due to its ease of handling and controllable gel properties. In this study, we introduce calcium peroxide (CaO₂) as a H₂O₂-generating reagent to gradually supply a radical source for the HRP-catalyzed crosslinking reaction. This novel therapy can create stiff hydrogels without compromising the cytocompatibility of the hydrogels due to the use of initially high concentrations of H₂O₂. The physico-chemical properties of the hydrogels can be controlled by varying the concentrations of HRP and CaO₂. In addition, the controlled and sustained release of bioactive molecules, including H₂O₂, O₂, and Ca²⁺ ions, from the hydrogels could stimulate the cellular behaviors (attachment, migration, and differentiation) of human mesenchymal stem cells. Moreover, the hydrogels exhibited killing efficacy against both Gram-negative and Gram-positive bacteria, dependent on the H₂O₂ and Ca²⁺ release amounts. These positive results suggest that hydrogels formed by HRP/CaO₂ can be used as potential matrices for a wide range of biomedical applications, such as bone regeneration and infection treatment.