The four major serotype of dengue viruses as well as other members of the genus Orthoflavivirus are globally endemic. Diseases caused by orthoflavivirus infections are significant public health concerns; yet, to date, there are no approved antiviral treatments and limited vaccine options are available. Recent computational and in vitro studies have identified several genus-wide conserved genomic quadruplex forming sequences, suggesting that these complex RNA structures may have functional importance and therefore, offer new therapeutic targeting opportunities. This dissertation describes a structure-guided approach for identifying orthoflavivirus RNA quadruplex-targeting small molecules as an antiviral development strategy. Here, we report a high-resolution X-ray crystal structure of the WNV NS5-B quadruplex forming sequence that folds into a two-stack tetrad and is further stabilized by novel capping features and loop base pairing interactions. Subsequent bioinformatic analyses revealed that the NS5-B quadruplex sequence is highly conserved across the genomes of the four DENV serotypes. The formation of these DENV NS5-B quadruplexes in vitro was confirmed by biophysical characterization. Binding studies revealed several quadruplex stabilizing heterocyclic diamidine compounds that can bind to the orthoflavivrus NS5-B quadruplexes at high affinities and are non-toxic in cell cultures. Overall, these data demonstrate a potential for developing viral RNA quadruplex targeting diamidines as broad-spectrum antiviral therapeutics.