A molecular-level study of the influence of the alkyl chain length of quaternary ammonium ions (QAs) on the blocking action and the mode of binding with the bacterial KcsA K+-ion channel is carried out by molecular dynamics (MD) simulations as well as quantum mechanics/molecular mechanics (QM/MM) methods. The present work unveils distinct modes of binding for different QAs, due to differences in size and hydrophobicity. The QAs bind near the channel gate as well as at the central cavity, leading to a possible dual-site blocking action. Small-sized tetraethylammonium (TEA) and tetrabutylammonium (TBA) ions enter inside the channel cavity in the open state of KcsA but bind strongly in the closed state. TEA binds to the polar hydroxyl group of threonine residues situated at the channel gate via nonclassical H-bonding interaction (C-H center dot center dot center dot O), while TBA binds to a second binding site, the central cavity, with hydrophobic benzyl and sec-butyl side chains of phenylalanine and isoleucine residues via alkyl-pi and hydrophobic interactions (C-H center dot center dot center dot H-C). On the contrary, large tetrahexylammonium (THA) and tetraoctylammonium (TOA) ions bind the hydrophobic side-chain methyl and isopropyl of alanine and valine at the channel gate both in the open and closed states, thereby restricting the free movement of large QAs toward the center of the cavity. However, the binding to the hydrophobic benzyl and sec-butyl side chains of phenylalanine and isoleucine residues in the closed state is thermodynamically preferable. Also, the binding energy is found to increase with an increase in the alkyl chain length from ethyl (-16.4 kcal/mol) to octyl (-65.5 kcal/mol), due to an almost linear increase in dispersive interaction.