Thiadiazolines (TDZs) are pentagonal heterocyclic compounds derived from thiosemicarbazones (TSCs). TDZs have exhibited promising biological effects as chemotherapeutic drugs, leading to the possibility that they could also prove efficacious against different forms of cancer. This is supported by the anti-tumor properties of various TSCs, their chemical precursors. To explore the anti-tumor effects of four TDZs (B20, B21, B23, and B24) on breast cancer, experiments were conducted using human and murine breast cancer cell lines. The cytotoxicity of the compounds was assessed through MTS assay to determine IC50 values after 48 hours of treatment. It was observed that hormone-dependent cell lines were more responsive to B20 and B23 (both substituted with chlorine in positions 3 and 4 of the aromatic ring) compared to B21 and B24, while triple negative cell lines showed higher IC50 values for all the TDZs (LM05 Mix B20: 3.3M, B21: 44M, B23: 8.2M, B24: 17.2M; MCF-7 B20: 25M, B21: 270M, B23: 33.2M, B24: 40M; 4T1 B20: 380M, B21: undefined, B23: 74.6M, B24: 58.5M, LM3 B20: 54.9M, B21: undefined, B23: 41.8M, B24: 64.4M and MDA-MB-231 B20: 176.7M, B21: undefined, B23: 141.8M, B24: 52.3M). Furthermore, through the Ethidium Bromide and Acridine Orange staining technique, we found that the cytotoxicity of B20 and B23 on hormone-dependent cell lines was due to apoptosis. Additionally, the ability of LM05 Mix and MCF-7 cells to form colonies was reduced under the treatment of B20 and B23 compared to control cells (LM05 Mix B20: 36±6% and B23: 50,4±0.2%, MCF7: B20: 33±10% and B23: 43±12%, p˂0,05). Moreover, the cell cycle analysis revealed that treatment with B20 and B23 led to a G2 phase cell cycle arrest in sensitive cells. Overall, these findings suggest that B20 and B23 have promising anti-tumor effects on hormone-dependent breast cancer cells and warrant further investigation into their mechanism of action as potential therapies for breast c