Water in biodiesel emulsified fuel improves air�fuel mixing and reduces combustion temperature, thereby lowering nitrogen oxides (NOx) emissions in diesel engines. Studies on emulsified fuel formation often use chemical surfactants. However, biosurfactants, which are natural and eco-friendly, remain unexplored. Additionally, the common-rail fuel injection system increases the pressure, temperature, and shear forces of emulsified fuel, thereby altering its properties and emulsified fuel droplet size and distribution. This study investigates the influence of biosurfactant concentration (1�3 ) and fuel injection system pressures (34.5, 69.0, and 103.5 MPa) on emulsified fuel properties. We utilised microscopy and a customized MATLAB image-processing code, along with a hot-plate technique and high-speed camera, to analyze droplet size distribution and micro-explosion behaviours. The findings demonstrate that increasing the concentrations of biosurfactants from 1 to 3 leads to an increase in the density by 1.07 and viscosity by 10.81 but lower surface tension by 16.499 in emulsified fuel, subsequently reducing particle size, delaying micro-explosion, and lowering peak temperature. On the other hand, all fuel properties undergo a notable reduction after passing through the fuel injection system, with increases in fuel pressure and injection through the nozzle's orifices. In the case of fresh emulsion, with initial values of 841 kg/m3 density, 2.35 mm2/s viscosity, and 26.9 mN/m surface tension, these properties decreased to 829 kg/m3, 2.05 mm2/s, and 25.23 mN/m respectively, under an injection pressure of 103.5 MPa due to increasing the heating and shear forces generated by the fuel injection system while particle size distribution decreased from 2-4 µm to 1�2 µm from the fuel tank to the injector spray. The micro-explosion time of droplets from the fuel tank and injector spray operated at 34.5 MPa, 69.0 MPa, and 103.5 MPa was found to be 1.72 s, 1.97 s, 2.21 s, and 2.34 s, respectively. Correspondingly, the peak temperatures recorded were 272 °C, 277 °C, 282 °C, and 288 °C respectively. © 2024 Elsevier Ltd