Previous research has demonstrated that glyphosate efficacy is strongly influenced by root invading soil-borne microorganisms. However, this interaction among plants, glyphosate, and soil microorganisms has only been studied in a number of crop plants, but not in weed species. This is surprising since the soil biotic environment has a strong impact on the activity of this important herbicide. Gaining a better understanding of these interactions will shed more light on the performance of glyphosate in the field and the evolution of glyphosate-resistance. Therefore, the objective of this research was to evaluate the role of soil microorganisms in the evolution of glyphosate-resistance in three problematic weeds of the midwestern United States: giant ragweed (Ambrosia trifida L.), horseweed [(Conyza canadensis (L.) Cronq.], and common lambsquarters (Chenopodium album L.). Through a series of greenhouse and lab experiments we determined that root colonization by soil microorganisms increased the activity of glyphosate in glyphosate-resistant (GR) and -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, giant ragweed susceptibility to Pythium spp. did not differ among biotypes, in the absence of glyphosate. Utilizing next-generation sequencing revealed that the dynamics of the rhizosphere microbial community of GR and GS giant ragweed biotypes were altered in response to a glyphosate treatment. The results of this research demonstrate that rhizosphere interactions are fundamental to the mode of action of glyphosate, in select weed species. 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.