Members of the mitis group streptococci are the most abundant inhabitants of the oral cavity and dental plaque. Influenza A virus (IAV), the causative agent of influenza, infects the upper respiratory tract, and co-infection with Streptococcus pneumoniae is a major cause of morbidity during influenza epidemics. S. pneumoniae is a member of mitis group streptococci and shares many features with oral mitis group streptococci. In this study, we investigated the effect of viable Streptococcus oralis, a representative member of oral mitis group, on the infectivity of H1N1 IAV. The infectivity of IAV was measured by a plaque assay using Madin-Darby canine kidney cells. When IAV was incubated in growing culture of S. oralis, the IAV titer decreased in a time- and dose-dependent manner and became less than 100-fold, whereas heat-inactivated S. oralis had no effect. Other oral streptococci such as Streptococcus mutans and Streptococcus salivarius also reduced the viral infectivity to a lesser extent compared to S. oralis and Streptococcus gordonii, another member of the oral mitis group. S. oralis produces hydrogen peroxide (H2O2) at a concentration of 1–2 mM, and its mutant deficient in H2O2 production showed a weaker effect on the inactivation of IAV, suggesting that H2O2 contributes to viral inactivation. The contribution of H2O2 was confirmed by an inhibition assay using catalase, an H2O2-decomposing enzyme. These oral streptococci produce short chain fatty acids (SCFA) such as acetic acid as a by-product of sugar metabolism, and we also found that the inactivation of IAV was dependent on the mildly acidic pH (around pH 5.0) of these streptococcal cultures. Although inactivation of IAV in buffers of pH 5.0 was limited, incubation in the same buffer containing 2 mM H2O2 resulted in marked inactivation of IAV, which was similar to the effect of growing S. oralis culture. Taken together, these results reveal that viable S. oralis can inactivate IAV via the production of SCFAs and H2O2. This finding also suggests that the combination of mildly acidic pH and H2O2 at low concentrations could be an effective method to inactivate IAV.