: Gamma irradiation is a promising terminal sterilization method for nanoparticle-based biomedical systems. However, its potential effects on the physicochemical properties and biological performance of multifunctional nanomaterials must be carefully evaluated. This study aimed to assess the structural integrity, sterility, and cytocompatibility of magnetic nanoparticles (MNPs) and bioactive-glass-coated magnetic nanoparticles (MNPBGs), both based on magnetite (FeO), after gamma irradiation. MNPs and MNPBGs were synthesized and subjected to gamma irradiation at 25 kGy, with additional doses explored in preliminary evaluations. Physicochemical characterizations were performed using XRD, TEM, SAED, and Raman spectroscopy. FTIR analyses were conducted on bioactive glass (BG) controls without magnetite. Sterility was evaluated via microbiological assays. Cytocompatibility and nitric oxide (NO) production were assessed using RAW 264.7 macrophages and Saos-2 osteosarcoma cells. Prussian blue staining was used to evaluate cellular uptake. Gamma irradiation preserved the crystal structure, morphology, and size distribution of the nanoparticles. FTIR revealed only minor changes in the silicate network of BG, such as reduced intensity and slight shifting of Si-O-Si and Si-O-NBO bands, indicating limited radiation-induced structural rearrangement without affecting the material's stability or cytocompatibility. Microbiological assays confirmed complete inhibition of microbial growth. All irradiated samples exhibited high cytocompatibility, with MNPBGs demonstrating enhanced biological responses. Notably, MNPBGs induced a more pronounced NO production in macrophages. Cellular uptake of nanoparticles by Saos-2 cells remained unaffected after irradiation. Gamma irradiation at 25 kGy is an effective sterilization strategy that maintains the structural and functional integrity of MNPs and MNPBGs. These findings support their safe use in sterile biomedical applications, particularly for bone-related therapies involving immunomodulation and drug delivery, with potential relevance for cancer treatment strategies such as osteosarcoma.