To identify new potential anti-influenza compounds, we isolated six flavonoids, 2′-hydroxyl yokovanol (1), 2′-hydroxyl neophellamuretin (2), yokovanol (3), swertisin (4), spinosin (5), and 7-methyl-apigenin-6-C-β-glucopyranosyl 2″-O-β-d-xylopyranoside (6) from MeOH extractions of Ohwia caudata. We screened these compounds for antiviral activity using green fluorescent protein (GFP)-expressing H1N1 (A/PR/8/34) influenza A-infected RAW 264.7 cells. Compounds 1 and 3 exhibited significant inhibitory effects against influenza A viral infection in co-treatment conditions. In addition, compounds 1 and 3 reduced viral protein levels, including M1, M2, HA, and neuraminidase (NA), and suppressed neuraminidase (NA) activity in RAW 264.7 cells. These findings demonstrated that 2′-hydroxyl yokovanol and yokovanol, isolated from O. caudate, inhibit influenza A virus by suppressing NA activity. The moderate inhibitory activities of these flavonoids against influenza A virus suggest that they may be developed as novel anti-influenza drugs in the future.

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