Ilex paraguariensis or yerba mate (YM) is native from South America with regional importance. This study evaluates YM extract obtention and its antimicrobial activity. YM leaves were obtained from: (1) municipality of Cascavel - PR; and (2) of Santa Maria do Oeste - PR. The YM were weighed (80 g) and extracted with ethanol: water mixture (70:30; 4 L). The extraction was ultrasound-assisted employing a bath, 70 ºC, 80% power, 37 kHz frequency for 60 min. The extract was filtered and spray drier (flow rate 0.5 L/h; 1.2 mm nozzle; inlet air 140 ºC). The minimum inhibitory concentration (MIC) against Klebsiella pneumoniae ATCC 10031, Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 43888 was evaluated by the colorimetric method by micro dilution with resazurin. The extracts were applying (100 L; 100.0-0.049 mg/mL) in 96-well microplates with Mueller-Hinton broth. Levofloxacin (LV; 64.0-0.031 μg/mL) was used as a positive control. The minimum bactericidal concentration (MBC) was determined by applying 10 μL of the wells in standard plate count agar (PCA). For Klebsiella pneumoniae the MIC was 3,13 (1), 12,5 mg/mL (2) and < 0.031 μg/mL (LV); the MBC was 12,5 (1), 12,5 mg/mL (2) and 4 μg/mL (LV). For Staphylococcus aureus the MIC was 3,13 (1), 12,5 mg/mL (2) and 0.125 μg/mL (LV); the MBC was 12,5 (1), 25,0 mg/mL (2) and 1 μg/mL (LV). For Escherichia coli the MIC was 6,25 (1), 12,5 mg/mL (2) and < 0.031 μg/mL (LV); the MBC was 12.5 (1), 25.0 mg/mL (2) and 2 μg/mL (LV). The results obtained are in the range described by Tonet. Employing a similar YM extraction process, he obtained a MIC of 10 mg/mL for Escherichia coli and Salmonella Typhimurium and 5 mg/mL for Staphylococcus aureus. This study demonstrates the suitability of YM extract obtention and its antimicrobial activity.