Research on immunotherapy in breast cancer treatment has recently gained importance. In this context, natural killer (NK) cells have been shown to kill cancer cells without affecting normal cells. Our study used the NK-92 cells that were stimulated with anti-CD226 antibodies (sNK-92) to increase their activity to target MDA-MB-231 triple-negative breast cancer cells. MCF-12A normal breast cells were used as the control in all experiments. The cytotoxic effects of NK-92 and sNK-92 cells on MDA-MB-231 cells were investigated using lactate dehydrogenase tests. The sNK-92 cells were more cytotoxic than NK-92 cells on MDA-MB-231 cells. In contrast, a significant cytotoxic change was not observed in MCF-12A cells cocultured with NK-92 and sNK-92 cells. An increase in granzyme B levels after coculturing with sNK-92 cells was investigated using the granzyme B enzyme-linked immunosorbent assay. The sNK-92 cells secreted more granzyme B than NK-92 cells against MDA-MB-231 cells. This increase was not observed in MCF-12A, indicating that sNK-92 cells specifically target cancer cells. In addition, immunostaining was used to investigate the synthesis level of BAX, CASP3, and CASP9 proteins to determine whether the observed cytotoxic effect was due to apoptosis. These proteins were synthesized more in MDA-MB-231 cells cocultured with sNK-92 than with NK-92 cells. However, no increase in their synthesis was observed in normal breast cells cocultured with NK-92 and sNK-92 cells. In conclusion, NK-92 cells stimulated with anti-CD226 antibodies secrete more granzyme B, resulting in a greater cytotoxic effect by inducing programmed cell death (apoptosis). The fact that the observed effects on breast cancer cells were not observed in normal breast cells indicates that sNK-92 cells specifically target breast cancer cells. These results indicate the potential use of CD226-stimulated NK-92 cells in immunotherapy.