The nicotinic acetylcholine receptor (nAChR) represents the prototype of ligand-gated ion channels. It is vital for neuromuscular transmission and an important regulator of neurotransmission. A variety of toxic compounds derived from diverse species target this receptor and have been of elemental importance in basic and applied research. They enabled milestone discoveries in pharmacology and biochemistry ranging from the original formulation of the receptor concept, the first isolation and structural analysis of a receptor protein (the nAChR) to the identification, localization, and differentiation of its diverse subtypes and their validation as a target for therapeutic intervention. Among the venom-derived compounds, α-neurotoxins and α-conotoxins provide the largest families and still represent indispensable pharmacological tools. Application of modified α-neurotoxins provided substantial structural and functional details of the nAChR long before high resolution structures were available. α-bungarotoxin represents not only a standard pharmacological tool and label in nAChR research but also for unrelated proteins tagged with a minimal α-bungarotoxin binding motif. A major advantage of α-conotoxins is their smaller size, as well as superior selectivity for diverse nAChR subtypes that allows their development into ligands with optimized pharmacological and chemical properties and potentially novel drugs. In the following, these two groups of nAChR antagonists will be described focusing on their respective roles in the structural and functional characterization of nAChRs and their development into research tools. In addition, we provide a comparative overview of the diverse α-conotoxin selectivities that can serve as a practical guide for both structure activity studies and subtype classification.