Tobacco seed, as agricultural waste, could become a significant raw material for biodiesel production. Biodiesel extracted from naturally renewable resources, such as vegetable oils, becomes more and more interesting as an alternative to fossil-fuel. Tobacco seed oil could be obtained by ordinary extraction (maceration), as well as the extraction according to Soxlet. Recently, ultrasonic extraction has been used for the better yield of extractive substances from the hard parts of plants. Maceration is a classic method of extraction in which hard vegetable material is being immersed into a solvent. In this study, we used whole and pulverized tobacco seed. The extraction was done with the use of unpolarized solvents: n-hexane and petrol ether. For the extraction of tobacco seed oil (Nicotiana tabacum L.), type Otlja, we applied the optimal conditions (seed pulverization, the type of solvent, temperature, extraction technique, seed:solvent ratio). This kind of oil is not suitable for application in diesel engines, and therefore it must be modified by the method of transesterification. For more efficient transesterification, it is recommendable for the oil to contain less than 2% of free fat acids. Tobacco seed oil with a higher content of free fat acids (more than 18%), must undergo the method of esterification of free fat acids first, in order to decrease the content to less than 2%, and after that to the method of base catalyzed transesterification, by which the yield of about 91% of methyl-esters is obtained. Biodiesel obtained in this way has the fuel properties which are within the limits issued by the latest European (DIN EN 14214) standards. Due to decreased resources and high prices of fossil fuel, biodiesel has got growing interest. Biodiesel is a plant-derived fuel; it is biodegradable, with higher oxygen and without sulfur content. The use of biodiesel has also many environmental benefits: less greenhouse effect, less pollution air, water and soil and less health risk, compared to the use of fossil fuels. Currently, rapeseed oil, sunflower seed oil, soy bean oil and palm oils dominate world-wide biodiesel production (Korbitz, 1999). Many different non edible oils have already been used, such as castor seed oil (De Oliveira et al., 2004), rubber seed oil (Ikwuagwu et al., 2000), walnut oil (Foidl et al., 1996), tobacco seed oil (Giannelos et al., 2002) and tomato seed oil (Giannelos et al., 2005). One way of reducing the biodiesel production costs is to use the less expensive feedstocks containing fatty acids such as non edible oils, animal fats and oils, recycled or waste oil and byproducts of the refining vegetable oils (Mittlebach et al., 2004). Tobacco seeds are not commercial products, they are not collected from fields and they are a by-product of tobacco leaf production. The oil content of tobacco seed has been found to be in the range from 33-40% (Eshetu, 2000), with trilinolein and palmitodilinolein being the main triglycerides accounting for about 90% of the composition of the oil (Gofur et al., 1993). Main fatty acids in the seed oil are oleic acid, stearic acid, palmitic acid and linoleic acid. This oil contains a high content of cholesterol (Harris, 1999), but it is free from nicotine (Pater et al., 1998). Tobacco seed oil has physical and chemical properties that are comparable to current European specifications for diesel fuel and can be an appropriate substitute in a raw (Giannelos et al., 2002) and chemically modified form (Usta, 2005). There are several techniques for the extraction of oil from tobacco seed. Many factors affect the efficacy of extraction, such as: extraction technique, degree of pulverization of the vegetable material, solvent polarity, solid material and solvent ratio and temperature of extraction.