Microbial as well as endogenous nucleic acids are recognized by a group of endosomal Toll-like receptors TLR3, TLR7, TLR8 and TLR9. Recent discoveries significantly improved our understanding of molecular mechanism of their activation and their physiological role. Those include recognition of dsRNA through two nucleic acid binding sites of TLR3 ectodomain, activation of TLR9 by phosphodiester backbone of ssDNA, independent of the nucleotide sequence and phosphorothioate modified bonds, and the role of proteolysis in activation of TLR9. In addition, proteins that chaperone nucleic acids, such as HMGB1 or LL-37, have been described to mediate TLR activation. There is growing evidence that supports involvement of endosomal TLRs in a number of autoimmune diseases, suggesting a therapeutic potential of immunomodulatory endosomal TLR ligands. So far, inhibitory nucleic acids against TLR7, TLR8 and TLR9 as well as small compounds targeting downstream signal transduction of single or several endosomal TLRs have been reported. TLR-targeting drugs have been included in clinical trials as vaccine adjuvants or as therapeutic agents for the treatment of diseases, ranging from cancer, infections, asthma and allergy to autoimmune diseases.