Gramicidin S (GS) and Gramicidin C (GC), two linguistically similar but structurally unrelated membranotropic peptides, exhibit comparable cytotoxic and neuromodulatory activities while operating through distinct membrane-disruptive mechanisms. GS is a cyclic decapeptide forming a β-hairpin structure, whereas GC is a linear pentadecapeptide adopting a membrane-spanning β-helix conformation that forms head-to-head dimeric ion channels. Using artificial phospholipid bilayers and isolated rat brain synaptosomes, we comprehensively analyzed their biophysical behavior and neurochemical effects. Solid-state 2H and 31P NMR spectroscopy and differential scanning calorimetry (DSC) revealed that GS induces membrane thinning and promotes positive curvature stress, while GC causes bilayer thickening and induces negative curvature in fluid-phase phospholipid membranes. In planar lipid bilayers (BLM), GC formed long-lived, discrete dimeric channels independent of the BLM composition, while GS produced short-lived, polymorphic conductance events, consistent with ill-defined oligomeric pores only in certain lipid mixtures. Both peptides induced Ca²⁺ influx in mammalian cells via acid-sensitive ion channels, P2X4, and acetylcholine receptors, with GC showing selective activation patterns and GS causing broader, likely non-specific permeabilization. Synaptosomal assays revealed that GS caused a full release of [14C]glutamate and [3H]GABA at high concentrations, while GC triggered only partial release, plateauing at submaximal levels. Both peptides caused membrane depolarization and ATP release, and possess hemolytic potential and similar cytotoxicity across prokaryotic and eukaryotic cells. These findings demonstrate that while GS and GC share functional outcomes, their membrane interactions and bioactivity profiles deviate due to their distinct structural and mechanistic properties.