Erbium oxide nanoparticles (ErO-NPs) have attracted significant attention for their unique physicochemical properties, including high surface area, biocompatibility, and stability. However, the impact of ErO-NPs on lymphoma cells (LCs) has not been explored, making this an innovative avenue for exploration. Therefore, the current study aimed to explore the influence of ErO-NPs on cell viability, genomic and mitochondrial DNA integrity, reactive oxygen species (ROS) generation and apoptosis induction in human U937 LCs. ErO-NPs were characterized using X-ray diffraction (XRD) and transmission electron microscopy (TEM). The effect of ErO-NPs on cell viability and genomic DNA integrity was estimated after 48 h using the WST-1 cytotoxicity and alkaline Comet assays, respectively. The generation level of reactive oxygen species (ROS) and mitochondrial membrane potential were also analyzed. Flow Cytometry was used to assess apoptosis induction and quantitative RT-PCR was conducted to measure the apoptotic (p53), anti-apoptotic (Bcl2), and mitochondrial (ND3) gene expression. Our results demonstrated the purity and well distribution of ErO-NPs and revealed that ErO-NPs induce strong cytotoxicity on U937 cells, as evidenced by a concentration-dependent reduction in cell viability with an IC50 value of 3.20 µg/ml. Exposure of U937 LCs to the IC50 concentration (3.20 µg/ml) of ErO-NPs promoted excessive ROS generation, leading to dramatic damage to genomic DNA and mitochondrial membrane potential, as well as marked dysregulation of apoptotic (p53), anti-apoptotic (Bcl2) and mitochondrial ND3 gene expression. This cascade of events triggered both apoptosis and necrosis in ErO-NPs-treated U937 LCs. In conclusion, these findings highlight the strong in vitro cytotoxic potential of ErO-NPs against highly aggressive U937 LCs, mediated by excessive ROS production, which leads to severe genomic DNA and mitochondrial membrane damage, as well as profound alterations in apoptotic, anti-apoptotic and mitochondrial gene expression. Future research is needed to further investigate the potential use of ErO-NPs in treating lymphoma and to optimize their therapeutic efficacy.