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As organic tobacco is a consumer choice, it is necessary to explore the proper fertilization program with environmentally friendly practices. Α field experiment lasting 3 growing seasons established at the Agricultural University of Athens in order to examined the effect of comparison between organic and inorganic fertilization on (flue-cured) Virginia tobacco plants (Nicotiana tabacum L.) and soil. A randomized complete block design with four treatments (manure, compost, NPK and control) and three replications was set up. The results of analysis of variance for treatments showed that the organic matter of soil did not affected by the fertilization while CaCO3 significantly affected by fertilization. The N concentration in soil does not significantly affected by fertilization. Plants height was higher under the inorganic fertilization in comparison to that of the organic fertilization treatments. The final yield was affected by year and it is higher in control treatment with highest value 7,053.42 kg ha-1 The fertilization does not significant affect the uptake N leaves was affected by fertilization and was higher under inorganic fertilizer, ranged from 2.73 to 0.88%. The nicotine and sugar on Virginia tobacco were not significant affected by fertilization. A strong correlation was observed between yield and reducing sugars (r= 0.66). The results of the current study revealed that the organic fertilization affected certain soil characteristics and not the quality characteristics of tobacco.