The article presents the test results of a four-stroke, four-cylinder, naturally aspirated, DI 60 kW diesel engine operating on diesel fuel (DF) and its 5 vol% (E5), 10 vol% (E10), and 15 vol% (E15) blends with anhydrous (99.8%) ethanol (E). An additional ethanol–diesel–biodiesel blend E15B was prepared by adding the 15 vol% of ethanol and 5 vol% of biodiesel (B) to diesel fuel (80 vol%). The purpose of the research was to examine the influence of the ethanol and RME addition to diesel fuel on the start of injection and autoignition delay. The widely differing physical and chemical properties of the biofuel blends along with engine load and speed modes were taken into account to provide sound analysis of the experimental test results. Studies showed that the density of biofuel blends E5, E10, E15 and E15B was 0.33%, 0.65%, 0.95% and 0.56% lower at the temperature of 40 °C than the corresponding value (0.828 kg/m3) of diesel fuel. Kinematic viscosity of biofuel blends E5, E10, E15 and E15B also decreased by 7.8%, 11.0%, 13.0% and 10.8% at the temperature of 40 °C and the cetane number was 3%, 9%, 14% and 12% lower, respectively, compared to commercial diesel fuel. The use of biofuel blends E15 and E15B the autoignition delay increased by 4.4% and 9.5% compared to normal diesel operation at full pe = 0.67 MPa (100%) load and 1400 rpm speed at which maximum torque occurs.