This paper deals with the investigation of the influential parameters of a mathematical spray breakup model using different fuels. Beside injection system measurements, fuel physical properties and injection process characteristics were measured, because they are necessary for the spray simulation input. For validation purposes, spray was injected into motionless air at atmospheric pressure and room temperature and filmed with a highspeed camera. Spray macrocharacteristics have been determined on the recorded images. Using the simulation program, the injection processes for diesel, biodiesel, and their 50% blend B50 have been simulated. Spray mathematical model parameters were tuned based on the experimentally gained results. Primary breakup model parameters showed the biggest impact on the spray characteristics and were therefore expressed using the fuel physical properties, the injection process characteristics, and the working regime parameters. Spray simulations into the combustion chamber were made in the end. All of these results are presented and discussed in this paper.