The synthetic value of the C≡C bond has prompted assiduous efforts to develop effective methods for the synthesis of acetylenes. First introduced in the 1990s and then rediscovered twenty years later, alkynylbenziodoxol(on)es (EBXs) have rapidly emerged as one of the most powerful classes of alkynylating reagents. Easily accessed, bench stable and nontoxic, these compounds were initially employed for acetylene transfer onto nucleophilic substrates under a variety of conditions. However, they have later shown their great utility in the alkynylation of radicals. Key to their success is their cyclic structure, which provides them with enhanced stability and permits the fine-tuning of their reactivity through structural adjustments of the benziodoxol(on)yl core. After presenting the principal procedures for the preparation of EBXs, this review will summarize the most important synthetic applications of these reagents. The functionalization of small molecules is discussed first; following that, the more recently established methods for the labeling of biomolecules with EBXs are described.