The aim was to evaluate the influence of the degree of quaternization of N-trimethyl chitosans (TMCs) on the mucoadhesive and penetration enhancement properties towards buccal mucosa. Fluorescein isothiocyanate dextran (MW 4400 Da) (FD4) was used as model molecule. TMCs, obtained from chitosans of different MW (1460 and 580 kDa, respectively), were hydrated in distilled water and in pH 6.4 phosphate buffer (simulating the buccal fluid). The polymer solutions were subjected to mucoadhesion measurements towards bovine submaxillary mucin dispersion and porcine buccal mucosa and to FD4 permeation tests through porcine cheek epithelium. The trimethylation of chitosan allows maintenance or improvement of the mucoadhesive properties of the starting chitosans dependently on quaternization degree. In particular, the mucoadhesive properties increase on increasing degree of quaternization. The trimethylation does not produce any change in chitosan penetration enhancement properties when the medium is distilled water while if pH 6.4 buffer is used, the trimethylation produces an improvement in chitosan penetration enhancing effect. TMC derived from the lower MW chitosan and characterized by the highest degree of quaternization shows the best mucoadhesive and penetration enhancement properties and is the most promising TMC to improve the bioavailability of hydrophilic and large MW molecules (like peptides and proteins) when administered via buccal route.