In this study biodiesel was produced from waste sunflower cooking oil and ethanol using N,N′,N″-tris(3-dimethylaminopropyl)-guanidine as new catalyst, which allows to avoid unwanted side-reaction of saponification. The influence of process variables on the conversion was investigated using experimental design and response surface methodology. Independent factors were varied in the following range: molar ratio of alcohol/oil from 3:1 to 15:1; catalyst concentration from 0.5 to 3 wt%; reaction temperature from 40 to 80 °C; and reaction time from 40 to 200 min. An empirical model of the process was developed. With this model and the goals of minimizing the independent factors in the tested range and maximizing the conversion, the optimization resulted in the following conditions: 1.13 wt% of catalyst, a molar ratio of 11.16:1, temperature of 67.96 °C, and a reaction time of 80 min. With those optimal conditions, the conversion predicted by the model was 94.6%. •Biodiesel production from waste cooking oil and bioethanol with guanidine catalyst.•Process optimization for: temp., time, wt% catalyst, and molar ratio of reactants.•The optimal conditions were: oil/alcohol = 1/11.16, temp. = 68 °C, wcatalyst = 1.13 wt%.•At optimal conditions the conversion was 94.6%.