Eurycoma longifolia is a popular folk medicine in South East Asia. This study was focused on saccharide-containing compounds including saponins, mainly because of their medical potentials. Different organic solvents such as ethyl acetate, butanol, and chloroform were used to fractionate the phytochemical groups, which were consequently precipitated in cold acetone. Solvent fractionation was found to increase the total saponin content based on colorimetric assay using vanillin and sulfuric acid. Ethyl acetate fraction and its precipitate were showed to have the highest crude saponins after acetone precipitation. The samples were shown to have anti-proliferative activity comparable with tamoxifen (IC50 = 110.6 µg/mL) against human breast cancer cells. The anti-proliferative activities of the samples were significantly improved from crude extract (IC50 = 616.3 µg/mL) to ethyl acetate fraction (IC50 = 185.4 µg/mL) and its precipitate (IC50 = 153.4 µg/mL). LC-DAD-MS/MS analysis revealed that the saccharide-containing compounds such as m/z 497, 610, 723, 836, and 949 were abundant in the samples, and they could be ionized in negative ion mode. The compounds consisted of 226 amu monomers with UV-absorbing property at 254 nm, and were tentatively identified as formylated hexoses. To conclude, solvent fractionation and acetone precipitation could produce saccharide-containing compounds including saponins with higher anti-proliferative activity than crude extract against MCF-7 cells. This is the first study to use non-toxic solvents for fractionation of bioactive compounds from highly complex plant extract of E. longifolia.