Although buccal permeation investigations with antiretroviral drug solutions have confirmed their transbuccal delivery potential, studies on their formulation into delivery systems are lacking. Multipolymeric monolayered films (MMFs) with drugs and polymers of opposing solubilities will offer several advantages for the controlled release delivery of didanosine (DDI) via the buccal route. The aim of this study was to employ a co-blending-co-plasticization technique for preparation of MMFs containing Eudragit® RS 100 (EUD) and Hydroxypropyl methylcellulose (HPMC) and to undertake molecular modelling and in vitro characterizations. Uniform drug content (91–105%) with low variability was obtained for all films. Co-blending of DDI:HPMC:EUD (1:1:10) was required to achieve controlled drug release. The buccal permeability potential of DDI from the MMFs was successfully demonstrated with a permeability coefficient of 0.72 ± 0.14 × 10−2 cm/h and a steady state flux of 71.63 ± 13.54 μg/cm2 h. Films had acceptable mucoadhesivity (2184 mN), mechanical strength (0.698 N/mm2) and surface pH (6.63). The mechanism inherent to the mucoadhesive and drug release profile performance of the MMFs was elucidated via static lattice molecular mechanics simulations wherein a close corroboration among the in vitro–in silico (IVIS) data was observed. These extensive physico-mechanical and molecular atomistic studies have confirmed the use of MMFs containing DDI, HPMC and EUD as a buccal delivery system.