The aim of this study was to develop novel transdermal films of ondansetron HCl with high molecular weight chitosan as matrix polymer and 2-(2-ethoxy-ethoxy) ethanol (Transcutol (R)) as plasticizer. In this context, firstly the physicochemical properties of gels used to formulate transdermal films were characterized and, physicochemical properties and bioadhesiveness of the transdermal films prepared with chitosan gels were assessed. The impact of three different types of terpenes, namely limonene, nerolidol and eucalyptol on in vitro skin permeation of ondansetron from transdermal films were also examined. ATR-FTIR measurements were performed to investigate the effects of the chitosan film formulations on in vitro conformational order of stratum corneum intercellular lipids after 24 h permeation study. The results showed that the chitosan gels consisting of Transcutol (R) as plasticizer and terpenes as penetration enhancer may be used to prepare transdermal films of ondansetron due to the good mechanical properties and bioadhesiveness of the transdermal films. Eucalyptol (1%) showed higher permeation enhancer effect than the other terpenes and control. ATR-FTIR data confirmed that finding in which eucalyptol induced a blue shift in the both CH2 asymmetric and symmetric absorbance peak positions indicating increased lipid fluidity of stratum corneum.