The use of renewable biodiesel fuel in diesel engines can reduce the demand for depleting fossil fuels and reduce harmful emissions to the environment. In this research, an engine simulation is conducted using ANSYS Forte software, which allows for visualization of the spray inside the combustion chamber. The results show that biodiesel has higher liquid and vapor penetration lengths, higher droplet mass and diameter, and a longer breakup length. Molecular images of fuel molecules show that the temperature of biodiesel molecules is 141 °C lower than diesel molecules at 709 degree crank angle (°CA). These characteristics result in an extended evaporation time for biodiesel, consequently leading to poorer performance. Additionally, increased penetration length can lead to carbon deposits inside the combustion chamber. Therefore, such inefficiencies of biodiesel spray properties lead to lower combustive performance than diesel. In terms of performance, on average, biodiesel produces 16.9% lower power and 19.9% higher brake specific fuel consumption. On average, the emissions of CO, CO2, and HC of biodiesel are 17.8%, 3.41%, and 23.5% lower and NOx is 14.39% higher than the corresponding values obtained for pure diesel, respectively. In-cylinder combustion analyses show that the peak pressure of biodiesel is 0.5 MPa lower, the peak cycle temperature is 36 °C lower, the ignition delay is 4 °CA longer, the peak heat release rate is 16.5 J/deg. higher, and the combustion duration is 5.96 °CA longer compared to diesel combustion.