Abstract Tobacco seed oil has been evaluated as a feedstock for biodiesel production. In this study, all properties of the biodiesel that was produced from tobacco seed oil were examined and some solutions were derived to bring all properties of the biodiesel within European Biodiesel Standard EN14214 to verify biodiesel quality. Among the properties, only oxidation stability and iodine number of the biodiesel, which mainly depend on fatty acid composition of the oil, were not within the limits of the standard. Six different antioxidants that are tert-butylhydroquinone, butylated hydroxytoluene, propyl gallate, pyrogallol, α-tocopherol and butylated hydroxyanisole were used to improve the oxidation stability. Among them, pyrogallol was found to be the most effective antioxidant. The iodine number was improved with blending the biodiesel produced from tobacco seed oil with a biodiesel that contains more saturated fatty acids. However, the blending caused increasing the cold filter plugging point. Therefore, four different cold flow improvers, which are ethylene–vinyl acetate copolymer, octadecene-1-maleic anhydride copolymer and two commercial cold flow improvers, were used to decrease cold filter plugging point of the biodiesel and the blends. Among the improvers, the best improver is said to be octadecene-1-maleic anhydride copolymer. In addition, effects of temperature on the density and the viscosity of the biodiesel were investigated.