Currently available treatments for acute myeloid leukemia exhibit side effects that limit their use, with primary and secondary resistance as persistent issues. While edible mushrooms possess nutritional value, they are also an excellent source of bioactive compounds that may have the potential to treat multiple disease states. The aim of the present study was to investigate the in vitro inhibitory effects of chromatographic fractions from the methanol extract of Cantharellus cibarius in a human acute myeloid leukemia (AML) cell line MV4-11. The mushrooms were purchased from a grocery store, the fruiting bodies were triturated and then extracted with 99.9% methanol to generate a crude extract. This crude extract was then redissolved in methanol, filtered over cotton to remove insoluble solids, and then fractionated over Sephadex® LH-20. Each fraction was dried and then analyzed by reverse-phase HPLC. A typical UV wavelength was selected for the detection of possible anticancer compounds in C. cibarius based on the major chromophores in main fungal anticancer agents reported. Based on chromatographic profiles, specific fractions that might contain potential anticancer agents were combined, and the inhibitory activity of the combined fractions was assessed against the MV4-11 leukemia cell line. Data confirmed that one combined fraction LH-20F-IV showed the greatest degree of in vitro inhibitory activity against the selected cell line. Fast determination of the potential anticancer compound containing fraction was completed through the application of reported chemoinformatics on HPLC detection of possible chromophores in potential anticancer agents, in combination with cellular bioassays.