Due to its higher oxygen content, biodiesel fuel could play a vital role in the achievement of emission control in the heavy-duty transportation sector. Waste cooking oil (WCO) obtained from various sources such as the food industry, restaurants, and sewers could provide sustainable means of producing low-carbon fuel such as biodiesel. In this research, WCO biodiesel and conventional diesel fuels were characterized fundamentally in terms of their spray and gas entrainment qualities under diesel-like engine conditions using laser-induced fluorescence and particle image velocimetry (LIF-PIV). The impact of fuel injection parameters such as injection pressure and nozzle diameter on the fuel's spray and gas entrainment characteristics were investigated. Furthermore, an empirical equation was used to determine the droplet sizes of the sprays at different injection conditions. For both fuels, spray breakup and atomization were enhanced with the droplet size decreasing as injection pressure increased from 100 to 300 MPa and as nozzle size decreased from 0.16 to 0.08 mm. As a result of higher viscosity and surface tension, it was observed that WCO biodiesel produced longer spray penetration and narrower spray angle than diesel fuel. Furthermore, the quantity of gas entrained by WCO biodiesel spray was lower. As a result of an increase in the surface area, the percentage of gas entrained at the side periphery of the spray increased as time proceeded while the percentage of gas entrained via the spray tip decreased. The combined effect of ultra-high injection pressure of 300 MPa with a smaller nozzle hole diameter of 0.08 mm was observed to enhance gas entrainment processes.