Various honeys have been extensively studied for their growth inhibition and apoptosis-inducing potentials on cancer cells. Compared to other chestnut honeys, Sinop region chestnut honey has recently received a geographical indication certificate due to its purity and high nutritional content. In the present study, honey samples of the SINATE brand chestnut honeys produced in the Beekeeping Application Area of Sinop University Natural Products Application and Research Center (Turkey) were used. Randomly selected samples were characterized physicochemically and investigated for their antiproliferative effects against lung cancer cells (A-549), cervix cells (HeLa), human breast cancer cells (MCF-7), and healthy human embryonic kidney cells (HEK-293) by MTT test. Also, its antioxidant characteristics, LDH release, DNA fragmentation, and cell cycle arrest potentials against MCF-7 cells were determined. Physicochemical analyses showed that the refractive indexes of the samples ranged from 15.8% to 17.4%, which is within the limit recommended by the international standard for honey. HMF value was not detected in the Sinop chestnut honey samples, and the fructose/glucose ratio was found to be 1.23. Other physicochemical properties (moisture, pH, electrical conductivity, and proline amount) were within the standard. Cytotoxicity analysis showed that the honey was more effective against MCF-7 cells (IC50 = 10.43 ± 2.32 μg/mL) than HEK-293 (IC50 = 62.98 ± 18.24 μg/mL), HeLa (IC50 = 68.95 ± 11.21 μg/mL), and A-549 (IC50 = 171.48 ± 22.71 μg/mL). In further studies on MCF-7 cells, it was seen that this cytotoxic effect was accompanied by LDH leakage from the cell membranes. Antioxidant/oxidant status of MCF-7 cells after honey treatment compared to the control showed changes and significant increases in oxidative stress levels. These results revealed that honey samples may trigger cytotoxicity through plasma membrane disruption in MCF-7 cells. Similarly, a significant increase in DNA fragmentation levels was detected after treatments. It is clearly demonstrated that cellular components were damaged and cancer cell viability was inhibited due to oxidative stress. Inhibitions in MCF-7 cell viability were supported by cell cycle arrest. This study reveals that Sinop chestnut honey can exert a selective cytotoxic effect against MCF-7, which are ER-positive breast cancer cells. These findings may be attributed to phenolic compounds categorized as phytoestrogens in honey, because these components have been shown to induce apoptosis by stimulating both ER-α/−β subtypes.