We compared the life cycle of rapeseed oil butyl esters (RBEs) made from waste rapeseed oil with that of RBEs made from conventional rapeseed oil. Chemical and biotechnological methods of production are considered. Sodium hydroxide was chosen as a catalyst for the chemical production method; for biotechnological production, the lipase lipozyme RM IM was chosen. The life cycle assessment, which was performed using the CML-IA baseline V 3.02/EU 25 method (SimaPro software), shows that using waste oil has a positive effect on all impact categories with the application of either biotechnological or chemical production methods. The use of RBEs from waste oil (using the biotechnological production method) led to a reduction of the following environmental impacts (compared with fossil fuel impacts): abiotic depletion up to 79.1%, acidification 38.8%, eutrophication 18.33%, global warming 77%, ozone layer depletion 90.12%, human toxicity 71.8%, fresh water aquatic ecotoxicity 9.01%, marine water aquatic ecotoxicity 56.9%, terrestrial ecotoxicity 0.191% (according to our results the impact of diesel on terrestrial ecotoxicity was small), and photochemical oxidation 36.8%.