The efficacy of treatments for oral ailments is often challenged by a low residence time of the conventional pharmaceutical formulations in the oral cavity. The residence time in the oral cavity could be improved by using bioadhesive formulations, such as preparations based on polysaccharides. This study describes the formulation and the evaluation of polysaccharide-based nanosystems as drug delivery systems addressed to the oral cavity. Nanoparticles based on chitosan, alginate or pectin were prepared through self-assembly by ionotropic gelation using oppositely charged crosslinkers (tripolyphosphate or zinc). Characteristics of nanoparticles at increasing crosslinker concentration provided the basis for selecting the most suitable formulations. The nanoparticles were tested for cytotoxicity against buccal cells (TR146) and for stability in a medium simulating pH, ionic strength, electrolyte composition and concentration of saliva. Alginate nanoparticles were the most stable in the salivary environment, while chitosan nanoparticles were the most cytocompatible. Alginate nanoparticles and pectin nanoparticles revealed possible cytotoxicity due to the presence of zinc. This knowledge is important in the early design of polymer-based nanoparticles for oral usage and for potential improving of the biocompatibility of the investigated nanoparticles with the oral environment.