Synthetic plastic polymers are causing considerable emerging ecological hazards. Starch-based biofilms are a potential alternative. However, depending on the natural source and extraction method, the properties of starch can vary, affecting the physicochemical characteristics of the corresponding casted films generated from it. These differences might entail morphological changes at the nanoscale, which can be explored by inspecting their surfaces. Potato ( Solanum tuberosum ) is a well-known tuber containing a high amount of starch, but the properties of the biofilms extracted from it are dependent on the specific variety. In this research, four Ecuadorian potato varieties ( Leona Blanca , Única , Chola , and Santa Rosa ) were analyzed and blended with different glycerol concentrations. The amylose content of each extracted starch was estimated, and biofilms obtained were characterized at both macroscopic and nanoscopic levels. Macroscopic tests were conducted to evaluate their elastic properties, visible optical absorption, water vapor permeability, moisture content, and solubility. It was observed that as the glycerol percentage increased, both moisture content and soluble matter increased, while tensile strength decreased, especially in the case of the Chola variety. These results were correlated to a surface analysis using atomic force microscopy, providing a possible explanation based on the topography and phase contrast observations made at the nanoscale.