Dibucaine (DBC) is one of the most potent long-acting local anesthetics, but it also has significant toxic side effects and low water solubility. Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) have been proposed as drug-delivery systems to increase the bioavailability of local anesthetics. The purpose of the present study was to characterize SLNs and NLCs composed of cetyl palmitate or myristyl myristate, a mixture of capric and caprylic acids (for NLCs only) plus Pluronic F68 prepared for the encapsulation of DBC. We intended to provide a careful structural characterization of the nanoparticles to identify the relevant architectural parameters that lead to the desirable biological response. Initially, SLNs and NLCs were assessed in terms of their size distribution, morphology, surface charge, and drug loading. Spectroscopic techniques (infrared spectroscopy and electron paramagnetic resonance, EPR) plus small-angle X-ray scattering (SAXS) provided information on the interactions between nanoparticle components and their structural organization. The sizes of nanoparticles were in the 180 nm range with low polydispersity and negative zeta values (-25 to -46 mV). The partition coefficient of DBC between nanoparticles and water at pH 8.2 was very high (>10). EPR (with doxyl-stearate spin labels) data revealed the existence of lamellar arrangements inside the lipid nanoparticles, which was also confirmed by SAXS experiments. Moreover, the addition of DBC increased the molecular packing of both SLN and NLC lipids, indicative of DBC insertion between the lipids, in the milieu assessed by spin labels. Such structural information brings insights into understanding the molecular organization of these versatile drug-delivery systems which have already demonstrated their potential for therapeutic applications in pain control.