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Two new flavonol glucuronides, quercetin 3-O-β-d-2″-acetylglucuronide (1) and quercetin 3-O-β-d-2″-acetylglucuronide methyl ester (2), along with four known flavonoids (3-6) were isolated from the whole parts of Rotala rotundifolia. The structures of 1 and 2 were elucidated by application of various spectroscopic methods, including 1D and 2D NMR techniques. Biological evaluation showed that all of isolated flavonoid compounds have potent anti-oxidative activities by DPPH and superoxide anion methods, and kaempferol (3) and quercetin (4) exhibited significant anti-HBV activity assayed by anti-HBsAg production. The HPLC fingerprint with photodiode array detection (HPLC-DAD) for quality control of R. rotundifolia partitioned EtOAc layer was also established.