An acetone extract of the leaves of Garcinia oblongifolia showed antiviral activity against enterovirus 71 (EV71) using a cytopathic effect inhibition assay. Bioassay-guided fractionation yielded 12 new prenylated benzoylphloroglucinols, oblongifolins J-U (1-12), and five known compounds. The structures of 1-12 were elucidated by spectroscopic analysis including 1D- and 2D-NMR and mass spectrometry methods. The absolute configurations were determined by a combination of a Mosher ester procedure carried out in NMR tubes and ECD calculations. Compared to ribavirin (IC50 253.1 μM), compounds 1, 4, and 13 exhibited significant anti-EV71 activity in vitro, with IC50 values of 31.1, 16.1, and 12.2 μM, respectively. In addition, the selectivity indices of these compounds were 1.5, 2.4, and 3.0 in African green monkey kidney (Vero) cells, respectively.