A field experiment was conducted during two successive seasons of  2012/2013 and 2013/2014 at Phytochemistry Department  and Farm of Applied Research Center of Medicinal Plants (ARCMP) affiliated to the National Organization for Drug Control And Research (NODCAR). Present work aimed to study the effect of inoculation Pimpinella anisum L. seeds with arbuscular mycorrhizal fungi (my) and/or microbein (mi) and/or foliar spray plants with thidiazuran (TDZ)  combine with chemical fertilizer at half or full dose of NPK on  the volume of the volatile oil of anise seeds, physiochemical investigation (Specific gravity of each essential oil and Refractive index of each essential oil), Compositional analysis by GLC of each essential oil percentage (linalol, estragole, alpha-terpenol, cis-anethole, trans- anethol and anisaldhyde) , total phenolic content and total flavonoid content of dry seeds. The results showed that inoculation anise (Pimpinella anisum L.) seeds with mixture of mycorrhizal and microbein at full dose of NPK gave the highest yield of the volatile oil of anise seeds , total phenolic content and total flavonoid content of dry seeds. While treated plants with biofertilizer (mycorrhizal and/or microbein) /or sprayed plants with TDZ combine biofertilizer at half or full dose of NPK didn't have any significant effect on physiochemical investigation and compositional analysis of volatile oil by GLC  as compared to the control.