Grape waste management is one of the main problems of winery industries, but, conversely, grape waste contains a high amount of polyphenols that might protect against human diseases related to oxidative stress, such as colorectal cancer. Therefore, the aim of this work was to investigate the antioxidant and antiproliferative activities of a grape waste extract obtained by supercritical fluid extraction. Because the beneficial effect of grape is related to its content of polyphenolic molecules, the extract was chemically characterized by high-performance liquid chromatography in order to assess its major bioactive components. The antioxidant activity of the grape extract was determined. The results showed that the grape extract presents a strong antiradical activity in the in vitro 2,2-diphenyl-1-picrylhydrazyl radical assay and protects against reactive oxygen species production in human colon adenocarcinoma cells (Caco-2). In contrast, the extract did not protect in the citronellal thermooxidation system and showed a weak protective action against lipid peroxidation in Caco-2 cells. The clonogenic assay and the cell cycle distribution analysis showed that the grape extract has a significant antiproliferative effect in a tumor cell line. These data indicate that grape extract is a promising product to be used as an anti-free radical agent and could exert a chemopreventive action.