Arracacha starch and potato starch are alternatives to synthetic polymers as starting materials for the production of biofilms. Herein, we determined the optimal concentrations of glycerol and potato or arracacha starch to obtain biofilms with high whiteness, transparency value (TV), and elastic modulus (E). Central composite design with the response surface methodology and desirability function are used to optimize the concentration of starch and glycerol. The biofilms are formed by the casting method using gelatinized starch suspensions (3-5%) mixed with glycerol (1-3%). The solubility and swelling power differences of the potato and arracacha starches indicated differences in the starch structures, which affected the properties of the biofilms. The potato and arracacha biofilms with the same starch and glycerol concentrations exhibited different TV and E. Linear effects, quadratics, and the interaction of the starch and glycerol have significant effects on the whiteness, TV, and E of the potato and arracacha biofilms (p < 0.05). The coefficient of variation for the obtained models is >0.98%. The optimal conditions determined using the desirability function are 5% starch and 1.02% glycerol for potato biofilms and 5% starch and 1% glycerol for arracacha starch biofilms.