It is known that the use of alternative fuels leads to changes in the operating parameters of internal combustion engines, and the nature of the changes in most cases is not known. Therefore, the question of researching the main operating indicators of the internal combustion engine supplied with RME B100 biodiesel fuel is important, and the results will help to eliminate or reduce negative factors that can lead to the deterioration of the operational and technical indicators of the internal combustion engine. The purpose of the research was to develop an experimental research facility using appropriate equipment and to study the main operational and technical parameters of the air-cooled D21A1 diesel engine on RME B100 biodiesel fuel. To reach the goal, the following tasks were formulated: the development of a test facility and research on the main technical and operational performance indicators of the D21A1 diesel engine on RME B100 biodiesel fuel. The authors’ previous research results were applied in the setting of the D21A1 test engine in the process of RME B100 biodiesel research; namely, to achieve maximum fuel combustion efficiency, the injection moment was increased by 6°. The results ensured the maximum efficiency of using RME B100 biodiesel in engines without making changes to the design of the latter. System analysis and the comparison method were used during the research. In the process of using RME B100 biodiesel fuel on the air-cooled D21A1 engine, we found a decrease in engine torque of 6.5%; a decrease in effective power of 6.7%; a growth in specific effective fuel consumption of up to 22.3%; and an increase in hourly fuel consumption of 14.1%. This is because the use of RME B100 fuel requires changes in the engine design that improve the mixture formation process.