Flavonoids play beneficial roles in various human diseases. In this study, a flavonoid library was employed to probe inhibitors of D-glycero-β-D-manno-heptose-1-phosphate adenylyltransferase from Burkholderia pseudomallei (BpHldC) and two flavonoids, epigallocatechin gallate (EGCG) and myricetin, have been discovered. BpHldC is one of the essential enzymes in the ADP-L-glycero-β-D-manno-heptose biosynthesis pathway constructing lipopolysaccharide of B. pseudomallei. Enzyme kinetics study showed that two flavonoids work through different mechanisms to block the catalytic activity of BpHldC. Among them, a docking study of EGCG was performed and the binding mode could explain its competitive inhibitory mode for both ATP and βG1P. Analyses with EGCG homologs could reveal the important functional moieties, too. This study is the first example of uncovering the inhibitory activity of flavonoids against the ADP-L-glyceroβ-D-manno-heptose biosynthesis pathway and especially targeting HldC. Since there are no therapeutic agents and vaccines available against melioidosis, EGCG and myricetin can be used as templates to develop antibiotics over B. pseudomallei.