The most common fuel used for powering compression ignition engines is diesel, whose main components are petroleum products. The constantly growing energy demand involves the implementation of new technical solutions and applying alternative fuels, including renewable ones, such as rapeseed oil, sunflower oil, peanut oil, and animal fats. The most commonly used biofuels are those obtained from chemically processed rapeseeds (transesterification) to provide them with physical−chemical properties similar to diesel fuel. The study presents the results of tests of a power unit fueled with different mixtures of diesel oil and fatty acid methyl esters. The experiment was carried out for a compression ignition engine of 81 kW power with direct fuel injection. Performance parameters of the vehicle power unit and its computer software were modified for the needs of the tests. Those modifications involved increasing the fuel dose and the fuel injection pressure. The test results were statistically analyzed. Based on the results, a simulation of power and torque was performed, depending on the vehicle computer system adjustment and the fuel mixture used. A simulation of the vehicle movement in a non-homogeneous environment (variable road conditions) was performed concerning the vehicle motion kinematics. The simulations were carried out in a V-SIM 5.0 program. The simulation was performed at five speeds, respectively: 0 km/h, 25 km/h, 50 km/h, 75 km/h, and 100 km/h. The simulation made it possible to determine speed, acceleration, time, and distance. The analysis shows that the highest acceleration of 3 m/s2 was obtained for the BIO50 mixture, regulation V. The longest road section needed to achieve the maximum speed (100 km/h) was recorded for the BIO10 mixture, regulation II. The simulation duration ranged from 17.9 s to 17.74 s, depending on the adopted variant.