Propolis and its extracts show a wide spectrum of biological activity. Due to the necessity to use high temperatures and high polarity in the eluent, the obtained extracts are depleted of active compounds. The new, cold separation method allows obtaining a qualitatively better product containing a number of chemical compounds absent in extracts obtained using high-temperature methods. The purpose of our study was to evaluate the biological activity of propolis extracts produced with the cold separation method in four female breast cancer cell lines: MDA-MB-231, MDA-MB-468, MCF-7, and T-47D. The results of the breast cancer cell viability were obtained using the MTT test. Propolis extracts at 75 and 80% showed similar cytotoxicity against cancer cells, with the polyphenol fraction 75% being slightly more negative for cells. Propolis extracts at concentrations of 50, 75, and 100 µg/mL significantly reduced cell viability. With the exception of the MDA-MB-231 line, cell viability was also decreased after incubation with a concentration of 25 µg/mL. Our results suggest that propolis extracts obtained with the cold separation method may be considered as promising compounds for the production of health-promoting supplements. Keywords: propolis; cold separation; breast cancer