The phenolic profile of Isabella grape (Vitis labrusca) offers beneficial properties to human health and makes it a functional food product. In order to better understand the phenolic compounds found in this grape variety and the biological effect they induce on breast cancer cells, an ultrasound-assisted extraction was carried out. During the extraction of polyphenols from Isabella grapes organically grown in Antioquia (Colombia), parameters such as frequency (33 kHz and 40 kHz), time and solvent were optimized to finally obtain a crude extract with antioxidant properties (Oxygen Radical Absorbance Capacity, ORAC: 293.22 ± 34.73 μmol of Trolox/g of sample), associated with a total polyphenol content (TPC) of 43.14 ± 5.00 mg GAE/g sample and a total anthocyanin content composed of 17.69 ± 2.59 mg of malvidin-3-glucoside/100 g of sample. MCF-7 breast cancer cells were treated with different concentrations of the optimized extract, and results show a decrease in cell viability related to mitochondrial membrane depolarization, ROS increase, and chromatin condensation. To determine the possible death induction mechanism, molecular docking was simulated to predict the molecular interactions between the most abundant phenolic compounds in Isabella grape and the main apoptosis-related proteins. The results obtained from in silico and in vitro experiments were consistent with each other, suggesting that the phenolic compounds found in Isabella grape can be considered potential adjuvant chemopreventive agents for the treatment of breast cancer.