One of the alternative fuels, originating from renewable sources, is biodiesel fuel, which is introduced in diesel engines without major construction modifications on the engine. Biodiesel fuel, by its physical and chemical properties, is different from diesel fuel. Therefore, it is expected that by the application of a biodiesel fuel, the characteristic parameters of the injection system will change. These parameters have a direct impact on the process of fuel dispersion into the engine cylinder, and mixing with the air, which results in an impact on the quality of the combustion process. Method of preparation of the air-fuel mixture and the quality of the combustion process directly affect the efficiency of the engine and the level of pollutant emissions in the exhaust gas, which today is the most important criterion for assessing the quality of the engine. The paper presents a detailed analysis of the influence of physical properties of a mixture of diesel and biodiesel fuels on the output characteristics of the fuel injection system. The following parameters are shown: injection pressure, injection rate, the beginning and duration of injection, transformation of potential into kinetic energy of fuel and increase of energy losses in fuel injection system of various mixtures of diesel and biodiesel fuels. For the analysis of the results a self-developed computer program was used to simulate the injection process in the system. Computational results are verified using the experiment, for a few mixtures of diesel and biodiesel fuels. This paper presents the verification results for diesel fuel and biodiesel fuel in particular.