In order to improve the production of biodiesel from an economic and environmental point of view, it is necessary to research the application of new catalysts, environmentally friendlier and cost-effective raw materials and their process optimization. Conventional catalysts, i.e. NaOH and KOH, when applied to vegetable oils with higher content of free fatty acids and water, usually lead to saponification. The resulting soap makes the separation of products difficult and thus increase waste water production. Furthermore, the use of edible vegetable oils has a negative impact on the food market by reducing the quantity of available farmland and increasing the price of food. Methanol is the most commonly used alcohol for transesterification. But it is toxic and produced from non-renewable sources, increasing greenhouse gas emissions. For this reason, the activity of the new guanidine catalyst N,N',N''-tris(3-dimethylaminopropyl)-guanidine (GV3) for the tansesterification of waste oil with various lower alcohols (methanol, ethanol, n-butanol and i-butanol) were examined. Ethanol is particularly interesting as a raw material because it can be produced from renewable sources, i.e. bioethanol. The process optimization with ethanol and waste oil was carried out for the most important process parameters (mass fraction of the catalyst, the molar ratio of ethanol and waste oil, reaction temperature and time) in accordance with the factorial central composite design of experiments. As result the significant empirical mathematical model was developed. Using Response Surface Methodology (RSM) optimal conditions were determined for the tested parameters. The goals of the optimization were to maximize the conversion, minimize the mass fraction of catalyst, at the lowest temperature and the shortest reaction time with the molar ratio of ethanol and waste oil closer to stechiometric. Depending on the importance given to a individual goal, different optimal conditions were calculated. The chosen optimal conditions were: the catalyst mass fraction of 1.72 wt.%; the molar ratio of ethanol and the waste oil 5.35:1; reaction time of 80 min at 40 °C with the calculated conversion of 91.44 %.