Antibacterial activity of yerba-mate (YM) and its industrial residue lacks deeper exploring. The objective of the work was to evaluate the antibacterial activity against pathogenic bacteria and polyphenolic content from YM leaves and twigs extracted for different periods. Staphylococcus aureus, S. epidermidis, Listeria monocytogenes and Escherichia coli were tested against aqueous extract from YM leaves and twigs and mode of action was evaluated by microscopy. Results showed that the aqueous extract of twigs inhibited both Staphylococcus species when extraction happened for 18h. The leaf extract showed inhibition against S. aureus. L. monocytogenes and E. coli were not inhibited. Extraction performed for 10 minutes did not yield antibacterial activity. No significant difference was observed in the concentration of total polyphenols or condensed tannins in the three evaluated extracts. Microscopy showed changes in the morphology of the S. aureus bacterial cell. YM and its byproduct is a promising source of functional compounds.