Despite all debates about its safe use, glyphosate still is the most widely applied active ingredient in herbicide products with renewed approval in the European Union until 2033. Non-target organisms are commonly exposed to glyphosate as a matter of its mode of application, with its broader environmental and biological impacts remaining under investigation. Glyphosate displays structural similarity to phosphoenolpyruvate (PEP), thereby competitively inhibiting the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), crucial for the synthesis of aromatic amino acids in plants, fungi, bacteria, and archaea. The majority of microbes, including the gut bacterium Escherichia coli (E. coli), possess a glyphosate-sensitive class I EPSPS, making them vulnerable to glyphosate's effects. Yet, little is known about glyphosate’s interactions with other bacterial proteins or its broader modes of action at the proteome level. Here, we employed a quantitative proteomics and thermal proteome profiling (TPP) approach, to identify novel protein binding partners of glyphosate in the E. coli proteome. Glyphosate exposure significantly altered amino acid synthesizing pathways, including increased abundance in shikimate pathway proteins, suggesting a compensatory mechanism. Extracellular riboflavin concentrations were elevated upon glyphosate exposure, while intracellular levels remained stable. Thermal proteome profiling indicated an effect of glyphosate on the thermal stability of certain proteins beyond the target enzyme EPSPS, including AroH and ProA. An elevated structural similarity between the substrates of the interaction candidates and glyphosate, similar to the competitive binding between PEP and glyphosate at the EPSPS, could be a reason for their interaction with the herbicide. Overall, glyphosate induced metabolic disturbances in E. coli, extending beyond its primary target, thereby providing new insights into glyphosate's broader impact on microbial systems.