Gut microbial β-glucuronidases show varied substrate specificity and inhibition propensity, highlighting the necessity of discovering broad-spectrum β-glucuronidase inhibitor to meet clinical needs. In this study, two prenylflavonoids sanggenon C (SGC) and kuwanon G (KWG) exhibited strong inhibition on p-nitrophenyl-β-D-glucuronide-hydrolyzing activity in pooled human gut microbiota and eight bacterial isolates, while amoxapine, a previously reported Escherichia coli β-glucuronidase (EcoGUS) inhibitor, only showed poor inhibition on the pooled samples and relatively selective inhibition on individual strains with incomplete inhibition on Staphylococcus.pasteuri 3I10. Both prenylflavonoids exhibited mixed-type inhibition against the recombinant enzymes EcoGUS and S. pasteuri 3I10 β-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 β-glucuronidase-mediated drug toxicity/efficacy warrants further investigation.