Tissue engineering and regenerative medicine (TERM) rely on advanced biomaterials and scaffolds that require strict sterilization without sacrificing their structural and functional properties. Conventional sterilization methods, including steam, ethylene oxide, and gamma irradiation, often compromise scaffold integrity, alter surface chemistry and/or leave toxic residues. Ozone (O₃) has emerged as a promising alternative sterilant because of its strong oxidizing potential, broad-spectrum antimicrobial activity, and residue-free decomposition. Importantly, ozone sterilization can preserve—and in some cases enhance—scaffold bioactivity by maintaining cytocompatibility and favorable surface chemistries that support cell adhesion and differentiation. This review critically evaluates the role of ozone sterilization in the context of TERM applications, focusing on its physicochemical properties, disinfection kinetics, material compatibility and regulatory perspectives. Evidence from studies on polymethyl methacrylate (PMMA) scaffolds, bone implants, and hydrogel-based systems suggests that, under optimized conditions, ozone can achieve high sterilization efficacy without significant degradation of mechanical or chemical properties. However, challenges related to process validation, health and safety considerations, and scalability remain. The review highlights opportunities for integrating ozone into automated biomanufacturing workflows and identifies key research gaps to support the broader adoption of ozone sterilization in TERM applications.