As the demand for fossil fuels has increased tremendously, cooking oil is found to be an effective source of biodiesel, but storage problems and NOX emissions are the major disadvantages. The current study discusses the effect of natural antioxidant additive Basil on the performance, emission and combustion characteristics of used cooking oil biodiesel in a diesel engine. The Basil powder prepared has been characterized through scanning electron microscopy, Fourier transform infrared spectroscopy, energy-dispersive spectrum and CHNS analysis. Thermal analysis of the prepared Basil particles has been carried out by employing thermal gravimetric analysis and differential scanning calorimetry. The addition of ethanol extracts of the antioxidant increased the oxidation stability measured in terms of the induction period of the biodiesel. The DPPH scavenging activity for the Basil extract increased with concentration, which represents the antioxidant characteristics. The composition of fatty acid and fuel properties of the prepared biodiesel have been determined by means of gas chromatography–mass spectrometry. The antioxidant was added to used cooking oil blend (UCOME20) at a concentration of 500, 1000 and 1500 ppm for which the stability has been determined using zeta potential. The results show that brake thermal efficiency and brake-specific fuel consumption reduced with the addition of antioxidants. NOx emission decreases, whereas hydrocarbon, carbon monoxide and smoke emissions increase. Also, maximum cylinder pressure and heat release rate decreased with the addition of Basil antioxidant to UCOME blend when compared to UCOME20.