Viral activation of toll-like receptors (TLRs) on dendritic cells (DCs) leads to production of various cytokines, including antiviral type I interferons (IFNs). Synthetic ligands specific for TLRs are also able to induce the production of type I IFNs (IFNα/β) by DCs, suggesting that these ligands have potential as antiviral drugs. In this in vitro study we extensively investigated the antiviral activity of various TLR ligands. Mouse bone marrow (BM) cells were differentiated into plasmacytoid and conventional DCs (pDCs and cDCs), stimulated with various TLR ligands and tested the antiviral abilities of collected supernatants in an in vitro herpes simplex virus type 1 (HSV-1) infection model. We observed a significant IFNβ-, (but not IFNα-) dependent reduction in HSV-1 infection when a mixed pDC/cDC population was stimulated with the TLR9 ligand CpG. In the absence of pDCs, TLR stimulation resulted in less pronounced antiviral effects. The most pronounced antiviral effect was observed when both DC subsets were stimulated with poly(I:C). A similar noticeable antiviral effect was observed when fibroblasts (L929 cells) were stimulated directly with poly(I:C). These poly(I:C)-mediated antiviral effects were only partially IFNβ-mediated and probably TLR independent. These data demonstrate that TLR ligands are not only able to produce type I IFN but can indeed act as antiviral drugs. In particular poly(I:C), which exerts its antiviral effects even in the absence of DCs, may become a promising drug e.g. to prevent respiratory infections by topical intranasal application.