Silver nanoparticles are widely used in industry, medicine and agriculture, but its environmental impacts, especially on the microbial flora of agricultural soils, have not been studied so far. Therefore, the study of the safety of these substances in terms of the effect on the microbial flora of water and soil resources to regulate mandatory criteria for producer, distributor and consumers is essential. In this regard, the present research attempts to investigate the interaction between the concentration and size of silver nanoparticles with bacterial microflora of cotton-grown soil. In this study, the effect of two commercial formulations of colloidal nanosilver, LS2000 and L2000 on bacterial flora of cotton rhizosphere soil was evaluated. Soil was treated with 0, 5, 10, 25 and 50 ppm of each formulation before seed planting. Samples were collected from rhizosphere soil of cotton at seedling, squaring and bolling stages. PCR–DGGE (Denaturing Gradient Gel Electrophoresis) was used to evaluate the bacterial diversity in the soils treated with different concentrations of nanosilver formulations. For this, the GC U968F/L1401R primer was used. The results of electrophoresis using DGGE on 8% gel of acryl amide containing a gradient of denaturing agents showed that the LS2000 formulation had a higher effect on the bacterial flora of the cotton rhizosphere in comparison with the L2000 formulation at all three stages of sampling. This effect was proportional to the concentration of both formulations. Also, the bacterial species affected by the L2000 formulation was not identical to those of the L2000 formulation and in each gel obtained from each sampling phase, different positions of DNA bands were observed. This suggests a different response of bacterial flora in exposure with these two formulations. In general, it can be concluded that the size and concentration of nanosilver particles are two important factors in the quantity and quality of the effect on bacterial flora and it is necessary to consider this issue in determining the standards of nanosilver use.