Iron oxide nanoparticles (IONPs) have the potential to be utilized in a multitude of fields, including biomedicine. Consequently, the potential health risks associated with their use must be carefully considered. Most biosafety evaluations of IONPs have focused on examining the impact of the material's distinctive physicochemical attributes. However, the specific attributes of individual cells are frequently disregarded, particularly under the oxidative stress conditions. This may result in an underestimation of potential risk and impede the clinical translation of IONPs. The present study thus sought to evaluate the potential cytotoxicity and underlying mechanisms of IONPs in a pathological state characterized by mild oxidative stress. A cell model of mild oxidative stress was initially established in vitro. Subsequently, a series of indicators, including cell viability, live/dead ratio, mitochondrial membrane potential, and oxidative damage, were measured to assess the cytotoxicity of IONPs. Finally, a series of ferroptosis regulators were used to elucidate the involvement of ferroptosis. Preincubation with IONPs resulted in a significant reduction in cellular viability, morphological degeneration, elevated numbers of dead cells, impaired mitochondrial function, and increased oxidative damage under mild oxidative stress conditions in vitro. The cytotoxic effects of IONPs under mild oxidative stress are largely dependent on ROS and iron ions and are strongly associated with ferroptosis, which is based on the effects of ferroptosis regulators. The present in vitro study indicated that IONPs are toxic to cells under mild oxidative stress, which is linked to ferroptosis.