This article examines opportunities of enzyme application during the production of biodiesel from microalgae oil by its transesterification in the mixture with mineral diesel fuel. The oil and mineral diesel fuel ratio in the reaction mixture enabled a yield of 7% ester content in the mixture. Effectiveness tests were conducted on seven industrial lipases, and Lipozyme TL IM was selected as the most effective lipase for further optimization. The process of algae oil transesterification with ethanol was optimized by applying response surface methodology. The interactions and impacts of the following independent variables on the transesterification yield were evaluated: ethanol and oil molar ratio, process duration, lipase content, and temperature. Optimum conditions were determined: a temperature of 30 °C, 13.26% biocatalyst (from the oil content), an ethanol and oil molar ratio of 4.54:1, and a process duration of 13 h. The transesterification yield of the product under the above conditions reached 98%.