Tolcapone is a nitrocatechol-based drug used as adjuvant therapy in Parkinson's Disease (PD). Despite being effective in managing PD-associated symptoms, its use in the clinics is limited by its highly hepatotoxic profile. Due to its low serum half-life and moderate oral bioavailability, frequent and high daily doses are necessary to obtain a significant therapeutic effect, which might enhance the risk of hepatotoxic events in patients. Hence, our study focuses on the development, characterization and early in vitro characterization of tolcapone-loaded nanostructured lipid carriers (NLCs) to improve tolcapone therapy. The newly designed NLCs, with sizes up to 120 nm, were prepared using the emulsion-solvent evaporation method with an entrapment efficiency (EE%) over 98%. Plus, NLCs were shown to be stable at different pHs and to have mucoadhesive properties. Cytotoxicity assays in HepG2 cells revealed that NLCs showed decreased cell viability, likely as a result of excessive intracellular lipid accumulation. Using Caco-2 and Caco-2/HT29-MTX co-culture models for permeability assays, the nanoformulations were capable of significantly transporting more tolcapone across the cell monolayers in comparison to free tolcapone without compromising cell monolayer integrity. Plus, the nanoformulations efficiently inhibited COMT in a cellular-based COMT inhibition assay using HepG2 cells. Early in vivo toxicological testing using Caenorhabditis elegans (C. elegans) demonstrated survival percentages > 88% after acute exposure to the nanoformulations in concentrations up to 100 µM. Together, the results obtained so far demonstrate that tolcapone-loaded NLCs have the potential to be a valid therapeutic option for the treatment of PD in the future.