Gut microbial β-glucuronidases (GUSs) inhibition is a new approach for managing some diseases and medication therapy. However, the structural and functional complexity of GUSs have posed tremendous challenges to discover specific or broad-spectrum GUSs inhibitors using Escherichia coli GUS (EcoGUS) alone. This study first assessed the effects of twenty-one dietary flavones employing three Loop 1-type GUSs of different taxonomic origins, which were considered to be the main GUSs involved in deglucuronidation of small molecules, on p-nitrophenyl-β-D-glucuronide hydrolysis and a structure-activity relationship is preliminarily proposed based on both in vitro assays and a docking study with representative compounds. EcoGUS and Staphylococcus pasteuri GUS showed largely similar inhibition propensities with potencies positively correlating with the total hydroxyl groups and those at ring B of flavones, while docking results revealed strong interactions developed via ring A and/or C. Streptococcus agalactiae GUS (SagaGUS) exhibited distinct inhibition propensities, displaying late-onset inhibition and steep dose-response profiles with most tested compounds. The α-helix in loop 1 region of SagaGUS which causes spatial hindrance but offers a hydrophobic surface for contacting with the carbonyl group on ring C of flavones is believed to be essential for the allosteric inhibition of SagaGUS. Taken together, the study with a series of flavones revealed varied preferences for GUSs belonging to the same Loop 1-type, highlighting the necessity of adopting multi-GUSs instead of EcoGUS alone for screening broad-spectrum GUSs inhibitors or tailoring the inhibition based on specific GUS structure.