Gut microbial beta-glucuronidases show varied substrate specificity and inhibition propensity, highlighting the necessity of discovering broad-spectrum beta-glucuronidase inhibitor to meet clinical needs. In this study, two prenylflavonoids sanggenon C (SGC) and kuwanon G (KWG) exhibited strong inhibition on p-nitropheny1-beta-Dglucuronide-hydrolyzing activity in pooled human gut microbiota and eight bacterial isolates, while amoxapine, a previously reported Escherichia coli beta-glucuronidase (EcoGUS) inhibitor, only showed poor inhibition on the pooled samples and relatively selective inhibition on individual strains with incomplete inhibition on Staphylococcus.pasteuri 3110. Both prenylflavonoids exhibited mixed-type inhibition against the recombinant enzymes EcoGUS and S. pasteuri 3110 beta-glucuronidase (SpasGUS). Molecular docking studies predicted phenolic groups of SGC and KWG as key structures interacting with the allosteric site of SpasGUS, while the phenolic hydroxyl (SGC) or benzopyranyl (KWG) group accounts for the hydrogen bond interaction with EcoGUS. The potentials of these broad-spectrum inhibitors in alleviating bacterial beta-glucuronidase-mediated drug toxicity/efficacy warrants further investigation.