Independent of the injury induced by influenza infection can promote bacterial superinfections remains unclear. Furthermore, it is unclear whether the phenomenon of influenza-mediated impairment of host defense is generalizable to other viral pathogens. Clinically, a number of viruses have been associated with bacterial co- or super-infections, including respiratory syncytial virus and rhinovirus, both of which are RNA viruses Therefore, we performed this study to test the hypothesis that simply activating viral RNA recognition receptors in the host respiratory tract would lead to impairment of bacterial clearance. We adopted an approach using synthetic compounds, specifically poly I:C, imiquimod, and gardiquimod, which are known to mimic the effects of viral nucleic acids on the GDC-0941 immune system, followed by bacterial challenge. Poly I:C is a synthetic compound that has been shown to activate TLR3, a receptor that recognizes dsRNA in the endosome, and the RIGlike receptors Retinoic-inducible Gene and Melanoma Differentiation-associated Protein 5 cytoplasmic receptors that recognize RNA viral nucleic acids. Imiquimod and gardiquimod activate TLR7, which recognizes single-stranded RNA. Both Poly I:C and TLR7 agonists are being considered as therapeutic or preventive agents for combating a variety of respiratory pathogens of pandemic or bioterrorism potential, including pandemic influenza, H5N1 avian flu, and Francisella tularensis, as they are believed to be an effective and safe “booster” of antiviral immune responses. In our murine model of pulmonary infection, we administered poly I:C or TLR7 agonist intranasally, followed by intratracheal challenge with common respiratory pathogens that cause postinfluenza bacterial pneumonia, to determine whether stimulation of antiviral immune pathways would increase susceptibility to secondary bacterial infection. We found that poly I:C exposure, similar to influenza infections, impairs clearance of S. pneumoniae and S. aureus. Moreover, the detrimental effects of poly I:C appear to be mediated by type I IFNs. Our findings suggest that poly I:C may not be a benign immunostimulatory molecule, and raises concern over its role as a preventive or therapeutic agent during viral pandemics. Viral infections are a clinically significant risk factor for bacterial pneumonia. Although influenza is widely recognized to predispose hosts to secondary bacterial pneumonias, epidemiologic studies have demonstrated that other respiratory viruses also appear to be associated with bacterial pneumonias. However, the mechanisms for this phenomenon are still poorly understood, and models testing various combinations of different viruses and bacteria are difficult to establish. Therefore, to circumvent this issue, we have adopted the approach of examining whether activation of antiviral pathways by administration of viral nucleic acid ligands common to respiratory RNA viruses could be employed as an approach to dissect out the pathways responsible for post-viral infectionmediated immune impairment. Furthermore, we wished to determine whether simply activating antiviral immune response pathways using viral RNA mimetics was sufficient to have deleterious effects on antibacterial host defense, and if so, which pathways appeared to be critical for this phenomenon. We found that exposure to the TLR3 and RIG-I ligand, poly I:C, was sufficient to impair pulmonary clearance of secondary bacterial infection, using two clinically-relevant gram-positive pathogens, S. pneumoniae and MRSA, as the second “hit”. There appeared to be a dose-dependent effect of poly I:C on the level of impairment.