The increase of waste generated by non-biodegradable materials combined with the difficulty in recycling most of the available synthetic packaging has encouraged the exploration of new materials, such as biodegradable films produced from natural polymers. The babassu (Orbignya phalerata) consists of epicarp, mesocarp, endocarp and almonds. The mesocarp represents 20% of the whole fruit and is an interesting source of starch, which makes a promising material for the production of biodegradable packaging. In this context, the aims of this work were the development of biodegradable films based on mesocarp babassu flour, a non-conventional source. The films were produced according to the casting method and the influence of glycerol concentration (20.0, 22.5, and 25.0 g/100 g flour) on the mechanical, barrier, and structure properties of babassu films has been evaluated. All films produced with glycerol presented homogeneous surface with no bubbles or cracks and good handling characteristics. The visual aspect was not affected by different plasticizer content and, in general, the film had a good appearance with reddish coloration that can be attributed to the reflection of the original color of the raw material used. Regarding the microstructure, the babassu films had a discontinuous structure with irregularities and small cracks, which may be related to the presence of more than one macromolecule in the polymer matrix (starch, protein, lipids, and fibers). Films features were affected by glycerol concentration. Babassu films had a decrease in tensile strength, Young’s modulus and water-resistance with rising plasticizer content from 20 to 25 g of glycerol/100 g of flour. Glycerol molecules have hygroscopic character and present high capacity to interact with starch chains, thereby enhancing the molecular mobility throughout the film structure. In general, babassu mesocarp seems to be a very promising material for the formulation of coatings and films.