Hyperuricemia (HUA), a prevalent metabolic disorder, needs new xanthine oxidase (XOD) inhibitor studies to reduce severe side effects. In this study, the inhibitory activities of 20 dietary flavonoids against XOD were determined in vitro, and a structure-activity relationship (SAR) model was subsequently developed. Diosmetin and quercetin emerged as the most potent inhibitors, both of which displayed mixed-type inhibition. SWISS-MODEL-based docking revealed that the key binding sites for diosmetin are Asn768 (active site) and Phe1009 (allosteric site), whereas those for quercetin are Lys771 (active site) and Phe914 (allosteric site). In hyperuricemic mice, diosmetin and quercetin reduced serum uric acid levels, suppressed XOD activity, and improved renal damage. Analysis by 16S rRNA sequencing revealed that both diosmetin and quercetin supplementation increased the abundance of beneficial bacteria, such as Lactobacillus johnsonii, and reduced the abundance of harmful bacteria, such as Desulfovibrio fairfieldensis. Notably, diosmetin shows lower potential hepatotoxicity than quercetin, as evidenced by the restoration of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in HUA-induced liver injury and a higher binding docking score with the p38MAPK protein. These findings provide a robust scientific basis for developing natural therapeutics with enhanced efficacy and safety.