Lepidium meyenii (maca) is an important food source renowned for its rich profile of bioactive compounds linked to antitumor and antioxidant activities. Herein, bio-guided fractionation and metabolomics were used to investigate L. meyenii potential to produce enriched-fractions with high antiglioma activity. Fractionation was performed by column chromatography using silica gel and Sephadex LH-20 resin as stationary phases, whereas cytotoxicity assays against C6 glioma cells were used as biomarker for the activity of the fractions. UHPLC-Q-TOF-MS/MS and chemometrics were used to assess the chemical profile of the fractions. Fractions F6.5.7 and F6.5.9 were the most potent, reducing C6 cell viability by 99% and 67%, respectively. Chemometrics revealed that these fractions were enriched in macamides and polyunsaturated fatty acids, including 9-oxo-10E,12E-octadecadienoic acid, linolenic acid, and macamide B. These findings highlight the potential of Lepidium meyenii as a functional food source for developing novel antiglioma agents through targeted fractionation of its bioactive compounds.