Continuous glyphosate use has contributed to an increasing number of problematic glyphosate-resistant (GR) weeds. The mechanism of resistance in many GR weeds is poorly understood, in part, due to a poor understanding of how exactly glyphosate kills a plant. In previous research conducted on crop species, the efficacy of glyphosate was shown to be greater in unsterile soils compared to sterile soils. Soil microorganisms were found to play an important role in glyphosate efficacy. The objective of this work was to determine the role of soil microbes in the resistance to glyphosate of three problematic weeds of the midwestern United States: giant ragweed (Ambrosia trifida L.), horseweed [(Conyza canadensis (L.) Cronq.], and common lambsquarters (Chenopodium album L.). A series of experiments were conducted to address glyphosate resistance from a unique approach; by investigating the interaction between weeds and soil microorganisms. Root colonization by soil microorganisms increased the activity of glyphosate in GR and glyphosate-susceptible (GS) biotypes of giant ragweed and a GS common lambsquarters biotype, but not in horseweed biotypes. The GS biotypes of each weed species were colonized by a greater number of soil microorganisms, specifically oomycete (e.g. Pythium spp. and Phytophthora spp.) pathogens, when treated with glyphosate, compared to the GR biotypes. However, utilizing next-generation sequencing revealed that the microbial community of the rhizosphere of GR and GS giant ragweed biotypes contained 98% Bacteria, and rarely identified Pythium spp. Glyphosate does cause changes to the diversity of the rhizosphere microbial community 3 days after treatment, and needs to be investigated further. The results of this research demonstrate that rhizosphere interactions are fundamental to the mode of action of glyphosate. These findings suggest that the range of tolerance to glyphosate observed in weeds and the evolution of resistance in weed biotypes may also be influenced by rhizosphere interactions. Understanding this interaction is crucial in understanding the biology of these weed species and resistance to this herbicide.