Glycerol-plasticized soy protein isolate (SPI) based films were prepared by compression with the aim to obtain environmentally friendly materials for packaging applications. Previously to the hot-pressed step, the protein was dispersed in water, the pH was fixed to values higher, lower and at the isoelectric point of SPI (pH=4.6), and the dispersion was freeze-dried. The effect of pH on physico-chemical properties has been explained using Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), and thermo-gravimetric analysis (TGA). The changes observed by FTIR in the intensity of the bands corresponding to the amide group showed that pH affected protein–glycerol interactions. Apart from pH effect, heat and pressure also affected the grade of denaturation of SPI shown by the disappearance of the DSC peak corresponding to 7S globulin. Mechanical properties were also evaluated and related to pH and storage time. Both tensile strength and elongation at break are higher at basic pHs, when the unfolding of protein seems to be optimum in order to interact with the plasticizer. Mechanical properties remained invariable after having been stored under specific conditions for two months. Preparation of SPI-based biofilms processed at different pHs by compression is an innovative study in this field, in which the most employed technique to prepare films has been casting.