The use of iron-nickel alloy nanoparticles (Fe-Ni ANPs) is increasing daily in various fields. People are increasingly exposed to these nanoparticles for occupational and environmental reasons. Our study determined some of the effects of Fe-Ni ANP exposure and impacts on human health at the cellular level. The cytotoxic and genotoxic potentials of Fe-Ni ANPs were investigated by XTT, clonogenic, comet, and GammaH2AX analyses using Beas-2B cells. Annexin V, multicaspase, and cell cycle arrest methods were used to understand the apoptotic mechanism of action. The intracellular ROS method was used to determine the primary mechanism that leads to cytotoxic and genotoxic activity. The Fe-Ni ANPs showed cytotoxic activity with the XTT and clonogenic methods: they had genotoxic potential, as demonstrated via genotoxicity methods. It was determined that the cytotoxic effect was realized by the caspase-dependent apoptotic pathway, and the cells were stopped at the G0/G1 stage by Fe-Ni ANPs. Increased intracellular ROS due to Fe-Ni ANPs led to cytotoxic, genotoxic, and apoptotic activity. Potential risks to human health due to Fe-Ni ANPs were then demonstrated at the cellular level.