The presence of residual pesticides in the soil affect the microbial Communities, as well the continuous use of pesticides exacerbates this problem. Glyphosate is one of the most used herbicides in the world. Up to date several studies have evaluated the tolerance and resistance of bacteria to glyphosate. Nitrogen-fixing bacteria play an important role in soil fertility; thus, the alteration of these bacterial communities decrease soil fertility. The objective of this study was to evaluate the effect of glyphosate application on four bacterial strains Pantoea agglomerans, Rhizobium nepotum, Rhizobium radiobacter, and Rhizobium tibeticum. Glyphosate was applied as the sole source of carbon at the rate (0 g/l, 0.5 g/l, 1g/l, 3g/l, 6g/l and 12 g/l) with two methods. Microbial growth was measured by the Colony Forming Units (CFUs /ml) method. Comparing with the control, our results showed that the growth of the four strains decreased by increasing the concentration of glyphosate. The four strains have shown resistance to glyphosate in the direct enrichment compared to the continued enrichment method. Comparing strains with each other, Rhizobium radiobacter is the most resistant strain to glyphosate.