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An organized approach has been made in this research to assess the diesel engine behaviour by fuelling novel biofuel. The unique and non-toxic, renewable citronella biofuel is extracted by steam distillation process. Moreover, extracted citronella oil exhibits similar viscosity to diesel. In this analysis, gas chromatography-mass spectrograph (GC–MS) and Fourier infrared (FT-IR) techniques were employed to characterize the chemical composition of citronella oil. The test fuel was prepared by mixing several blend ratios of citronella oil, which has been intermixed with diesel by volume basis, namely B20, B40, and B60. Its properties were appraised according to material standards. The test fuels were examined on a mono-cylinder conventional diesel engine. From the experimental exploration, it has been observed that citronella oil can succeed up to 20% at the top load condition in that order. Appreciably, the tailpipe Smoke emission has been reduced by 31% than a standard diesel operating mode, with a minor increase in oxides of nitrogen (NO X) and carbon dioxide (CO 2) emission by 12.5% and 7.1%. Besides that, carbon monoxide (CO) and hydrocarbon (HC) are 16.34% and 22.2% lesser than regular diesel fuel mode at the peak load condition. The BTE of citronella oil, along with its blend, exhibits minimal values when correlated with diesel fuel along with several combinations B20 blend, had better efficiency. The B20 (20% Citronella oil + 80% Diesel) claimed 1.47% lower in engine BTE and 2.7% higher fuel consumption than a diesel engine. The combustion analysis reported that citronella fuel had significant improvement in-cylinder pressure (CP), exhaust gas temperature (EGT), and heat release rate (HRR) with considerable reduction in ignition delay (ID). [ABSTRACT FROM AUTHOR]