G-quadruplexes (G4s) are noncanonical secondary structures that fold within guanine (G) rich strands of regulatory genomic regions. Recent evidences suggest their intimate involvement in important biological processes such as telomere maintenance, end-capping and protection, chromosome stability, gene expression, viral integration, and recombination. Mechanistic details of how and why G4 structures influence biological function indicate a rationale for treating G4s as emerging molecular targets for future therapeutics. In other words, the structural heterogeneity with well-defined binding sites, thermal stability and abundance of G4s in telomeres, oncogene promoter regions, and viral genomes make G4s attractive targets for small molecules, aimed to selectively recognize them over all other nucleic acids structures, particularly duplex forms that are most abundant in the genome. Herein, a critical survey of well-characterized G4-interactive ligands as potential tools in anti-cancer and antiviral therapies is presented. Effects that these ligands selectively exert in vitro and in vivo models are summarized. Unique ligands involved in specific G4 recognition are put forward. A key question, how to design and develop new G4 specific ligands that conform to the structural and physicochemical requirements for optimal biological activity, is discussed by considering both remarkable advances over the last few years and our recent contributions.