This research explores how varying glycerol concentrations (0–30 wt%) affect the physicochemical and mechanical characteristics of pectin films, derived from Citrus limon waste and enriched with lemon essential oil. The films were produced using the casting method. The findings show that glycerol significantly impacts film thickness, swelling behavior, water solubility, moisture content, water vapor permeability, and structural and mechanical characteristics. FTIR spectroscopy confirmed molecular interactions between glycerol and the film matrix. Notably, films with 20–30 wt% glycerol had reduced transparency. Mechanically, glycerol increased the elongation at break, enhancing flexibility, while a 5 wt% glycerol concentration optimized tensile strength. However, higher glycerol levels led to decreased tensile strength. Principal Component Analysis identified 5 wt% glycerol as optimal for balancing flexibility and structural integrity. Additionally, glycerol-plasticized films were more hydrophilic than the control. These results highlight glycerol’s crucial role as a plasticizer and the importance of precise concentration control in biodegradable film formulations.