Many unique chemical metabolites with significant antitumor activities have been isolated from Garcinia species and have become a leading hotspot of antitumor research in recent years. The aim of this study was to identify bioactive compounds from different plant parts (leaf, branch, stem bark, fruit, and seed) of G. xanthochymus through combining LC-MS-based metabolomics with cytotoxicity assays. As a result, 70% methanol seed extract exerted significant cytotoxic effects on five human cancer cell types (HL-60, A549, SMMC-7721, MDA-MB-231, and SW480). LC-MS-based metabolomics analysis was used, including principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), in order to identify 12 potential markers from seed extract that may relate to bioactivity. LC-MS guidance isolated the markers to obtain three compounds and identified new isopentenyl phloroglucinols ( 1 - 3 , named garxanthochin A-C), using spectroscopic methods. Among them, garxanthochin B ( 2 ) demonstrated moderate inhibitory activities against five human cancer cell types, with IC 50 values of 14.71~24.43 μM. These findings indicate that G. xanthochymus seed has significant cytotoxic activity against cancer cells and garxanthochin B has potential applications in the development of antitumor-led natural compounds.