Background: Breast cancer is a common nonskin cancer in females and rarely can happen in males. It is the second leading cause of cancer death among women. In spite of different methods of treatment for cancer, traditional medicines, especially herbal drugs, have a significant contribution for the preventions and cure of cancer due to their lesser side effects compared with other treatment methods. Objective: Cardiospermum halicacabum Linn. seeds oil was selected to evaluate their cytotoxic activities against various human breast cancer cell lines. Materials and Methods: In this study, the oil was extracted from the seeds of C. halicacabum Linn. and fractionated by column chromatography technique. All the obtained fractions were separated on bases of their chromatography similarity and the cytotoxicity was investigated by sulforhodamine B colorimetric (SRB) assay. Different types of human breast carcinoma cell lines, namely MCF-7, MDA-MB-231, and MDA-MB-435, were selected for cytotoxicity test and the active fraction was subjected to analysis by gas chromatography-mass spectrometry (GC-MS) for the identification of components present in the active seed oil fraction. Results: The study showed the cytotoxicity effect of fractions of the seed oil against MCF-7 breast carcinoma, with 50% growth inhibition value of <10 μg/ml. The GC-MS analysis of the most active fraction suggested the presence of active components that lead to cause of this effect. Conclusion: The results support the potential medicinal value of C. halicacabum L. seeds oil with inhibition of the proliferation of MCF-7 cells as an active antineoplastic agent that can be target for cancer therapy research.