Hypothiocyanite (OSCN-) is a natural component of human saliva and is produced by the lactoperoxidase (LPO)/thiocyanate/hydrogen peroxide (H2O2) system. OSCN- has been previously shown to exhibit antiviral activity against influenza viruses (IFV) A/H1N1/2009 and A/H1N2/2009 in vitro as well as antimicrobial and antifungal activities. We elucidated the antiviral activity of OSCN- against both IFV types A and B and the mode of its antiviral action. OSCN- was produced constantly at 900 ± 200 μmol/l in Na3PO4 buffer solution containing NaSCN and LPO in the presence of H2O2 as an original OSCN- solution. In a plaque reduction assay, IFV A/PR/8/34 (H1N1), A/Fukushima/13/43 (H3N2), B/Singapore/222/97, and B/Fukushima/15/93 were exposed to various concentrations of OSCN- for 0 to 30 min before adsorption to MDCK cells, and plaque formation was examined. OSCN- exhibited significant similar antiviral activities against all four viruses without cytotoxicity, and the EC50 values for them were from 57 ± 16 to 148 ± 27 μmol/l regardless of the exposure times. The exposure of MDCK cells to OSCN- before viral adsorption did not affect its anti-IFV activity (EC50: more than 450 μmol/l), but the exposure after viral adsorption affected it moderately (EC50: 380 ± 40 μmol/l). Moreover, the exposure of virus particles to OSCN- at 450 μmol/l did not affect the hemagglutinin activity of IFV in hemagglutination inhibition assay. These results suggest that the attachment of OSCN- to the viral envelope critically contributes to the mode of antiviral action of OSCN- without interfering with viral adsorption. Keywords: hypothiocyanite; influenza virus type A; influenza virus type B; lactoperoxidase; antiviral activity.