Biodegradable films of chia by-products (mucilage and protein-rich fraction (PF)) incorporated with clove essential oil (CEO) were obtained and characterized. The effects of polymer concentration (PC; 1.0–3.0 %, w/v) and CEO concentration (0.1–1.0 %, v/v) were evaluated as well as the pH (7–10), using a 23 factorial design with four central points. The films exhibited moisture values between 11.6 and 52.1 % (d.b.), which decreased (p < 0.05) with increasing PC and CEO. The thickness of the films increased (p < 0.05) with increasing PC. PC and pH influenced (p < 0.05) the lightness (L) and variation in color between red and green (a). The orientation of the color to yellow-blue hues (b) decreased significantly (p < 0.05) with increasing PC. Transparency was significantly lower and higher (p < 0.05) than PC and CEO, respectively. The film surface morphology was evaluated using atomic force miscrocope images, and thermogravimetric analysis (TGA) was performed to study the thermal stability of the films. The displacement and tensile strength were significantly lower (p < 0.05) at higher concentrations of CEO, this variable being the only one with a significant effect. The chemical composition of the films was confirmed utilizing Fourier transform infrared (FTIR) spectroscopy. The proportion of CEO added to the films had a significant influence on antimicrobial activity, inhibiting the growth of both Escherichia coli and Staphylococcus aureus.