In the current study, a self-nanoemulsifying drug delivery system (SNEDDS) containing N-trimethyl chitosan chloride (TMC) was developed and evaluated for its potential to deliver amphotericin B (AmpB) via the gastrointestinal tract. SNEDDS comprising Captex 355, Kolliphor RH40 and Propylene Glycol were optimized and characterized for their formation spontaneity, droplet size and drug loading. Investigations on the effects of the application SNEDDS containing TMC to a model intestinal epithelium (Caco-2 monolayer) indicated the capability of the formulations to induce the transient opening of tight junctions. Caco-2 cell viability studies confirmed the safety of the SNEDDS, whereas in vitro transport studies of AmpB through Caco-2 cell monolayers showed the permeation enhancing ability of TMC. Our results suggest that chitosan derivative TMC combined with a SNEDDS may be used as permeation enhancer to facilitate oral delivery of AmpB.