Bicontinuous microemulsions (B mu Es) are a promising biomembrane mimetic system for investigating the behavior of antimicrobial peptides (AMPs) and their delivery to open wounds to combat antibiotic-resistant microorganisms. The properties of the B mu E host are in turn affected by the guest AMP and can deviate from those of the unperturbed B mu Es, especially at higher AMP concentrations. Here we report the effect of an archetypal AMP, melittin, over a wide range of concentrations, on the nanoscopic dynamics of B mu Es formed by water/sodium dodecyl sulfate (SDS)/1-pentanol/dodecane, investigated using quasi-elastic neutron scattering (QENS). Two distinct motions are observed, namely, (i) the lateral motion of the surfactant on the surface of the oil channels and (ii) the internal motion of the surfactants. It is found that melittin restricts both the lateral and the internal motion, thereby acting as a stiffening agent. The lateral motion is more strongly affected, at low concentration of melittin. The lateral diffusion coefficient decreased sharply, approaching a constant value at higher melittin concentration. These results are in sharp contrast with the recent dynamic light scattering and neutron spin echo results which showed that at the length and time scales longer than those probed in the current work, melittin enhanced the long-range collective and local undulation motions of B mu Es. Considered together, our results indicate that incorporation of melittin modulates the dynamics differently depending on the spatial and temporal regimes, in which the dynamics are being probed. The addition of melittin at low concentrations increased the magnitude of the zeta potential, but further increase of the melittin concentration decreased it. This suggests that addition of melittin at low concentrations led to increase in the surfactant concentration, but did not affect the negative charge per surfactant molecule, while further addition of melittin led to ion pairing of melittin with the oppositely charged surfactant. This study therefore demonstrates how the addition of melittin hinders the lateral motion of surfactants as a result of the strong association between melittin and SDS, suggesting that the release of AMPs from B mu E-based delivery vehicles may be hindered. (C) 2021 Elsevier Inc. All rights reserved.