In this study, the antihyperuricemic effect of Acacia confusa heartwood extracts and their phytochemicals on potassium oxonate (PO)-induced acute hyperuricemia was investigated for the first time. All treatments at the same dosage (100 mmol/kg) were administered to the abdominal cavity of PO-induced hyperuricemic mice, and serum uric acid level was measured at 3 h after administration. In experimental mice, serum uric acid level was significantly suppressed by the administration of A. confusa heartwood extracts and their major phytochemicals, (-)-2,3-cis-3,4-cis-3,3',4,4',7,8-hexahydroxyflavan, (-)-2,3-cis-3,4-cis-4'-methoxy-3,3',4,7,8-pentahydroxyflavan, melanoxetin, transilitin, and okanin, relative to the PO group. The direct inhibitory effect of these five compounds on xanthine oxidase (XOD) activity was examined using isothermal titration calorimetry (ITC). Among them, melanoxetin showed a more remarkable inhibitory effect on XOD activity than allopurinol, a clinical drug used for XOD inhibitor. To further understand the stereochemistry between XOD and melanoxetin (or allopurinol), structure-based molecular modeling was performed. Melanoxetin undergoes extended interactions in the hydrophobic region via the 3',4'-dihydroxyphenyl moiety, thus accounting for its higher binding affinity to XOD than allopurinol. These results indicate that A. confusa heartwood extracts and their major phytochemicals exhibit strong XOD inhibitory effects, which reduce serum uric acid levels while inhibiting uric acid generation in purine metabolism.