The aim of this work was to investigate in vitro the transdermal permeation of caffeine from a new bioadhesive film, using rabbit ear skin as a barrier. The effects of film composition and of the presence of penetration enhancers in the formulation were studied. The obtained fluxes were compared with those shown by commercial formulations. The results obtained indicate that the bioadhesive film gave rise to a higher transdermal permeation compared to a commercial gel and to a saturated solution of caffeine in water. Additionally, the film did not present the typical time lag of solution and gel. Another peculiar feature of the film is that the percentage of permeated active substance is much higher than that obtained from commercial formulations. Finally, it was possible to modulate caffeine permeation from the film by adding different enhancers/solvents.