α-Conotoxins inhibit nicotinic acetylcholine receptors (nAChRs) and are used as probes to study cholinergic pathways in vertebrates. Model organisms, such as Drosophila melanogaster, express nAChRs in their CNS that are suitable to investigate the neuropharmacology of α-conotoxins in vivo. Here we report the paired nanoinjection of native α-conotoxin PIA and two novel α-conotoxins, PIC and PIC[O7], from the injected venom of Conus purpurascens and electrophysiological recordings of their effects on the giant fiber system (GFS) of D. melanogaster and heterologously expressed nAChRs in Xenopus oocytes. α-PIA caused disruption of the function of giant fiber dorsal longitudinal muscle (GF-DLM) pathway by inhibiting the Dα7 nAChR a homolog to the vertebrate α7 nAChR, whereas PIC and PIC[O7] did not. PIC and PIC[O7] reversibly inhibited ACh-evoked currents mediated by vertebrate rodent (r)α1β1δγ, rα1β1δε and human (h)α3β2, but not hα7 nAChR subtypes expressed in Xenopus oocytes with the following selectivity: rα1β1δε > rα1β1δγ ≈ hα3β2 > > hα7. Our study emphasizes the importance of loop size and α-conotoxin sequence specificity for receptor binding. These studies can be used for the evaluation of the neuropharmacology of novel α-conotoxins that can be utilized as molecular probes for diseases such as, Alzheimer's, Parkinson's, and cancer.