The unsaturated content of biodiesel makes it prone to oxidation resulting in variations in the fuel properties, hindering its widespread application. Variations in biodiesel properties impact injection, spray, evaporation, mixing and combustion processes. The present study investigates the effect of accelerated oxidized Karanja biodiesel on injector flow, macroscopic spray, and engine characteristics. The accelerated oxidation of Karanja biodiesel is carried out by heating and bubbling the air through the fuel. The variations in fuel properties that profoundly influence spray and engine characteristics are analyzed before and after accelerated oxidation. Even though biodiesel viscosity is increased beyond the ASTM specification limit due to accelerated oxidation, the variations in the density, surface tension, and calorific value are marginal. The injector flow and macroscopic spray characteristics are investigated for fresh and oxidized biodiesel using a constant volume spray chamber at different chamber and injection pressures. The results indicate a similar fuel flowrate and injection velocity for the fresh and oxidized biodiesels at identical test conditions. Under identical test conditions, the macroscopic spray characteristics between the test fuels are negligible. Engine experiments with fresh and oxidized biodiesel are carried out in an automotive truck diesel engine at rated torque speed and variable load conditions. A shorter ignition delay (∼20% lower), less intense premixed combustion, and lower nitrogen oxide (NOx) emissions (∼27% lower) are observed with oxidized biodiesel. The study concludes that despite significant variations in the kinematic viscosity of fresh and oxidized biodiesels (∼28% higher), the variations in macroscopic spray and engine performance characteristics are insignificant.