The contamination of water bodies by pesticides and antibiotics is a concerning environmental problem on a global scale. We investigated the impact of commonly used herbicides and antibiotics on bacterial isolates from the Guarani Aquifer System (GAS), the second largest aquifer in the world, in agriculture-intensive regions in southern Brazil. A total of 23 isolates were exposed to 2,4-D and glyphosate-based herbicides. Among these, 19 were tolerant (some presenting increased survival) to at least one herbicide. The collection site had a significant effect on isolates' maximum survival (MS), and a strong cross-tolerance between the two chemicals was detected, whereas seven out of 13 isolates (genera Bacillus, Lysinibacillus, Pseudomonas or Enterococcus) were resistant to at least one antibiotic. Isolates with the highest index of antibiotic resistance showed high MS values to herbicides, suggesting cross-resistance. We present the first characterisation of herbicide and antibiotic susceptibility of bacteria isolated from a deep aquifer. Herbicide tolerance was high and common, showing correlation with antibiotic resistance. The results suggest herbicides may impact microbial communities in aquifers, particularly concerning GAS-which spans four countries in South America-highlighting the importance of studying environmental microbes as potential remediators of contaminants, in line with the One Health principle.