Soy protein isolate (SPI) films plasticized with different contents of short and linear glycerol (G) and hyperbranched dendritic polyglycerol (DPG) in the presence of water were prepared for the first time with kneading and compression molding; these were analyzed in relation to their visual, morphological, microstructural, mechanical, and water-and oxygen-barrier properties. It was shown that the film prepared with a mixture of 15G15DPG (where the numbers represent the weight percentage of the respective compound) had a higher tensile strength (14.4%), lower elongation at break (85.7%), and improved water-barrier (54.6%) and oxygen-barrier (84.1%) properties compared to the SPI film plasticized only with 30G. The attenuated total reflectance-Fourier transform infrared spectra of the plasticized SPI films indicated that such properties were related to the approximately 11.3% higher conversion of SPI from the a-helical conformation to the intramolecular b-sheet structures for the 15G15DPG films. This resulted in finer films with lower surface roughnesses and surface areas. On the other hand, further increases in G and DPG revealed an opposite effect and worsened the properties; this was much more pronounced by the increased DPG amount because of SPI unfolding and aggregation and resulted in microporous films.