Mango seed kernel (MSK) has demonstrated to be a valuable source of bioactive phenolic compounds with antiproliferative activity against colon cancer cell lines. In this work, a phenolic MSK extract obtained by sequential pressurized liquid extraction (PLE) was submitted to supercritical antisolvent fractionation (SAF) to attain enriched fractions with improved inhibitory effect on HT-29 colon cancer cells. SAF was optimized using a Box-Behnken experimental design and considering as main factors: pressure, percentage of water in the feed extract and feed/sc−CO2 flow rate ratio. The highest inhibitory cell proliferation effect (70.51 ± 1.14%) was obtained in the extract using 50% of water v/v in the feed and 0.0625 feed/sc−CO2 ratio at 15 MPa. A targeted phytochemical profiling by LC-q-TOF-MS/MS followed by a multivariate statistical analysis of the observed bioactivity and the chemical composition evidenced the presence of characteristic compounds which might explain the enhanced antiproliferative activity of the optimal SAF extract.