BACKGROUND: The organophosphate pesticides have the potential to impact microbial diversity, but their influence on antibiotic resistance (AR) in bacteria remains understudied. OBJECTIVES: The objective of our study was to evaluate the impact of exposure to acetylcholinesterase inhibitors on glyphosate tolerance and AR in Gram-negative bacteria isolated from the digestive tracts of Thai farmers. METHODS: Human fecal samples from Thailand, grouped by pesticide exposure level measured by acetylcholinesterase blood concentration, were cultured on MacConkey (McK) agar with or without 7 g/L of a glyphosate-based formulation (GBF). Antibiotic susceptibility and glyphosate minimum inhibitory concentration (MIC) of isolated strains were assessed using the disk diffusion and broth microdilution methods, respectively. RESULTS: A total of 547 GNB were isolated from 112 human fecal samples. GBF medium predominantly selected Escherichia coli, Klebsiella pneumoniae, and Citrobacter freundii. GBF MICs ranged from 2 g/L to 16 g/L with K. pneumoniae species harboring the highest median MIC (16 g/L). AR rates were not significantly different between exposed and not exposed groups to pesticides. In contrast, six mobile colistin resistance (MCR)- and/or extended spectrum beta lactamase (ESBL)-producing E. coli strains were isolated from pesticide-exposed group, while only one colistin-resistant K. pneumoniae strain was isolated from a sample which was not exposed to pesticides. CONCLUSIONS: The results of our study underscore the need for further research, particularly on the impact of glyphosate exposure on colistin resistance and the prevalence of ESBL-producing strains. Additionally, we emphasize the importance of testing a broad range of pesticides to better understand their impact on AR.