Lactoperoxidase (LPO) is an enzyme found in several exocrine secretions including the airway surface liquid producing antimicrobial substances from mainly halide and pseudohalide substrates. Although the innate immune function of LPO has been documented against several microbes, a detailed characterization of its mechanism of action against influenza viruses is still missing. Our aim was to study the antiviral effect and substrate specificity of LPO to inactivate influenza viruses using a cell-free experimental system. Inactivation of different influenza virus strains was measured in vitro system containing LPO, its substrates, thiocyanate (SCN-) or iodide (I-), and the hydrogen peroxide (H2O2)-producing system, glucose and glucose oxidase (GO). Physiologically relevant concentrations of the components of the LPO/H2O2/(SCN-/I-) antimicrobial system were exposed to twelve different strains of influenza A and B viruses in vitro and viral inactivation was assessed by determining plaque-forming units of non-inactivated viruses using Madin-Darby canine kidney cells (MDCK) cells. Our data show that LPO is capable of inactivating all influenza virus strains tested: H1N1, H1N2 and H3N2 influenza A viruses (IAV) and influenza B viruses (IBV) of both, Yamagata and Victoria lineages. The extent of viral inactivation, however, varied among the strains and was in part dependent on the LPO substrate. Inactivation of H1N1 and H1N2 viruses by LPO showed no substrate preference, whereas H3N2 influenza strains were inactivated significantly more efficiently when iodide, not thiocyanate, was the LPO substrate. Although LPO-mediated inactivation of the influenza B strains tested was strain-dependent, it showed slight preference towards thiocyanate as the substrate. The results presented here show that the LPO/H2O2/(SCN-/I-) cell-free, in vitro experimental system is a functional tool to study the specificity, efficiency and the molecular mechanism of action of influenza inactivation by LPO. These studies tested the hypothesis that influenza strains are all susceptible to the LPO-based antiviral system but exhibit differences in their substrate specificities. We propose that a LPO-based antiviral system is an important contributor to anti-influenza virus defense of the airways.