Based on 3-levels-D-optimal design, involving as factors: methanol: oil, molar ratio; catalyst concentration, wt%; reaction time, min; and type of waste cooking oil, a statistical design of experiments strategy was performed to evaluate and investigate the biodiesel production process from different types of waste cooking oil using CaO prepared from waste eggshells. MATLAB software was employed for experimental design and data analysis. An empirical quadratic regression equation model was obtained describing the interrelationships between dependent and independent variables to maximize the response variable (biodiesel yield). The optimum values of the selected predictor variables were obtained by solving the quadratic model equation using LINGO software. They were found to be: methanol: oil, 9.15:1 molar ratio; catalyst concentration, 7.728 wt%; and reaction time, 75 min, regardless of the type of waste cooking oil used as the feedstock. The qualification and yield of biodiesel were comparable to those prepared using chemical CaO and immobilized standard enzyme Novozym 435.