Cotton is one of the important fiber plant grown worldwide commercially over 70 nations. India is the only country where all the cultivated species and certain hybrid combinations are cultivated commercially. Cotton occupies only five percent of the arable land and supports sixty million people having a direct bearing on the country’s economy. Cotton growers have so many constraints for cotton production but the most important is its high vulnerability to insects. Particularly to the larvae of Lepidopteran pests, which are impacting cotton production. During the entire growth period of Bt-cotton, Bt toxin is introduced into the soil through root exudates, decomposing plant material and/or pollen deposition. Deposition Bt toxin in soil may pose a potential risk to soil microorganisms such as plant growth promoting rhizobacteria, fungal population, Total bacterial population and arbuscular mycorrhizal (AM) fungi, a kind of non-target soil microorganisms fundamental for soil fertility and plant nutrition. It has been reported that Bt toxin leach out in the rhizosphere and inhibits the growth of soil microbes. Therefore, the present study was undertaken to study the impact of Bt-cotton and non-Bt-cotton cultivation on soil microflora at seedling stages of cotton plants in different sites of Jalgoan, Maharashtra. The obtained results revealed that Bt-cotton crop adversely affected the Arbuscular mycorrhizal population and percentage of root bit infection.