Escherichia coli and Staphylococcus aureus are globally among the most prominent bacterial strains associated with antibacterial resistance-caused deaths. Naturally occurring polyphenols, such as hydrolyzable tannins, have been shown to potently inhibit E. coli and S. aureus. The current study investigated the metabolome changes of E. coli and S. aureus cultures after treatments with different hydrolyzable tannins using an NMR metabolomics approach. Additionally, the effect of these tannin treatments influencing a more complex bacterial system was studied in a biomimetic setting with fecal samples inoculated into the growth medium. Metabolite concentration changes were observed in all three scenarios: E. coli, S. aureus, and fecal batch culture. The metabolome of E. coli was more altered by the tannin treatments than S. aureus when compared to control cultures. A dimeric hydrolyzable tannin, rugosin D, was found to be the most effective of the studied compounds in influencing bacterial metabolome changes and in inhibiting E. coli and S. aureus growth. It was also observed that the tannin structure should have both hydrophobic and hydrophilic regions to efficiently influence E. coli and S. aureus growth.