Zinc oxide nanoparticles (ZnO NPs) are increasingly applied in medicine, cosmetics, and environmental technologies, yet their interactions with blood cells remain poorly understood, raising cross-species safety concerns. Using frog (nucleated) and human (anucleate) erythrocytes as comparative models, we show that cellular architecture fundamentally shapes responses to ZnO NPs exposure. Human erythrocytes exhibited a dose-dependent progression from membrane deformation to eryptosis and hemolysis, reflecting the pronounced vulnerability of anucleate cells. In contrast, frog erythrocytes sustained nuclear DNA damage while largely preserving membrane integrity, highlighting the protective or reparative role of the nucleus. Molecular docking revealed energetically favorable interactions of ZnO NPs with ERα-LBD and DNA (ΔG = −4.28 and −5.68 kcal/mol, respectively), while quantum chemical analyses indicated electron-accepting properties and a narrow HOMO–LUMO gap, suggesting efficient macromolecular interactions and intracellular ROS generation. Together, these findings demonstrate that the presence of a nucleus shifts the primary target of nanoparticle toxicity from membrane to genome, providing novel mechanistic insights. This comparative study offers a robust framework for understanding nanomaterial reactivity across taxa and informs One Health-oriented risk assessments.