High Resolution Image Download MS PowerPoint Slide In this study, we investigated the biochemical mechanisms induced by magnetic nanoparticles (MNPs) in magnetically targeted photodynamic therapy (MT-PDT). An insight into the underlying oxidative/nitrosative stress mechanism is vital for optimizing treatment protocols, enhancing therapeutic efficacy, and minimizing adverse effects of MT-PDT. We have synthesized and characterized methylene-blue-loaded cobalt ferrite nanoparticles (MB-SCFNP) and evaluated their MT-PDT efficacy in breast cancer. Our in vitro cytotoxicity results revealed high cytotoxicity of MB-SCFNP in the presence of a static magnetic field + laser with an approximately 2-fold lower IC 50 value compared to laser only. Concurrently, minimal dark toxicity indicated the cytocompatible nature of MB-SCFNP. Moreover, MB-SCFNP-mediated apoptosis was found to involve the generation of reactive oxygen species (ROS). Furthermore, we observed enhanced intracellular ROS production under magnetic field + laser exposure. The biochemical assay results demonstrated the imbalance between ROS and nitrogen (RNS) species production and antioxidant defense mechanisms, which implicated the role of oxido-nitrosative stress in triggering cellular damage and cytotoxic effects. The observed changes in lactate dehydrogenase release, superoxide dismutase levels, and ROS production suggest oxidative stress, whereas an increase in nitric oxide levels indicates nitrosative stress. These cellular responses provide insights into the mechanisms of NP-mediated MT-PDT for breast cancer treatment.