In this research, response surface methodology (RSM) was applied to study the effects of Na-Montmorillonite (Na–MMT) and glycerol on some functional properties of corn starch films. Films were prepared by casting method according to central composite design (CCD). Originally image processing technique was used in order to estimate transparency of the films. It was found that Na–MMT absorbed UV light from 216 to 266nm. No antimicrobial activities were observed against Escherichia coli and Staphylococcus aureus. Contact angle analysis revealed hydrophilicity of starch films decreased utilizing nanoparticles and increased with plasticizer content. Increase in glycerol content decreased tensile strength sharply, while presence of Na–MMT increased it gently. The minimum of hydrophilicity and the maximum of tensile strength was obtained by maximization of clay and minimization of plasticizer in the considered range, which resulted in minimum transparency.