Toll-like receptors (TLRs) belong to a family of innate immune receptors that detect and clear invading microbial pathogens. Specifically intracellular TLRs such as TLR3, TLR7, TLR8 and TLR9 recognize nucleic acids such as double-stranded RNA, single-stranded RNA and CpG DNA respectively derived from microbial components. Upon infection, nucleic acid sensing TLRs signal within endosomal compartment triggering the induction of essential proinflammatory cytokines and type I interferons to initiate innate immune responses thereby leading to a critical role in the development of adaptive immune responses. Thus, stimulation of TLRs by nucleic acids is a promising area of research for the development of novel therapeutic strategies against pathogenic infection, allergies, malignant neoplasms and autoimmunity. This review summarizes the therapeutic applications of nucleic acids or nucleic acid analogues through the modulation of TLR signaling pathways.