Fatty acid-acylated cholines, a recently identified class of endogenous compounds, have been de tected in both human and animal organisms. Our prior work established that oleoylcholine (Ol-Chol), and other acylcholines, at micromolar levels, modulate the cholinergic system and are suitable as cationic lipids for introducing nucleic acids into human and animal cells. The present research examines the interaction with the nicotinic acetylcholine receptors (nAChR) of two ionic forms of Ol-Chol and two synthesized cat ionic lipids, each featuring a quaternary ammonium moiety and two oleic acid chains. A radioligand bind ing assay revealed that the affinity of acylcholines and synthetic cationic lipids for the muscle-type nAChR surpasses that for the human neuronal α7 nAChR by a factor of 2-5.5. Oleoylcholine iodide demonstrated a two-fold higher efficacy of mesylate in binding to the orthosteric site of muscle and α7 nAChR. In a func tional calcium imaging assay, both compounds exhibited superior inhibition of α7 nAChR by several orders of magnitude, suggesting potential interaction with allosteric binding sites. Compared to oleoylcholine, syn thetic cationic lipids demonstrated markedly reduced efficacy in binding to α7 nAChRs and, in contrast to oleoylcholine, induced a substantial cytotoxic impact on SH-SY5Y neuroblastoma cells, a phenomenon unaf fected by specific nAChR ligands. As a result, the nAChR-inhibitory properties are attributed to the quater nary ammonium group present in all studied compounds. However, the modification of the lipophilic moiety with two oleic acid residues curbs these properties but enhances cytotoxic activity through an alternative mechanism independent of nAChR.