A study of the injection process and spray behavior has been made for three different fuels. In particular, blends of rapeseed methyl ester (RME) with standard diesel fuel at 5% and 30% of biodiesel have been used for the current study, as well as pure RME.Hydraulic characterization of an 8-hole nozzle has been carried out using these three fuels, in order to explore and analyze the influence of fuel properties on mass flow rate and momentum flux at the nozzle exit. Additionally, spray visualization tests have been made in order to get information about spray cone angle, which allows the characterization of air-fuel mixing process. Finally, a theoretical derivation has been used to obtain further details of the microscopic characteristics of the spray and compare air-fuel mixing efficiency for the different biodiesel blends.