ABSTRACTThis work illustrates a comparative study on the applicability of the natural calcium oxide (CaO) prepared from waste eggshells and chemical CaO as basic heterogeneous catalyst in the transesterification of waste cooking oil (WCO) with methanol for production of biodiesel. Response surface methodology (RSM) based on D-optimal design of experiments was employed to study the significance and interactive effect of methanol-to-oil (M:O) molar ratio, catalyst concentration, reaction time, and mixing rate on biodiesel yield. Second-order quadratic model equations were obtained describing the interrelationships between dependent and independent variables to maximize the response variable (biodiesel yield) and the validity of the predicted models were confirmed. The activity of the produced natural biocatalyst was comparable to that of chemical CaO, producing high yield of biodiesel ≈91 and 98% at 8.57:1 M:O, 3.99 catalyst wt%, 31 min reaction time, and 398.88 rpm mixing rate at 60°C, respectively. Fuel properties of the produced biodiesel were measured and compared with those of Egyptian petro-diesel and international biodiesel standards. The overall biodiesel characteristics either prepared using natural or chemical CaO were comparable and acceptable, encouraging the application of CaO prepared from waste eggshells for production of biodiesel as an efficient, environmentally friendly, sustainable, and low cost heterogeneous catalyst.