Ilex paraguariensis is a native tree from South America known for its bioactive compounds. Processed leaves are consumed as hot and cold infusions, after process of harvesting (step 1), applying a flame bleach to inactive the enzymes (step 2), drying and milling the leaves (step 3). This study objective is to evaluate the processing steps impact on I. paraguariensis leaves composition and antimicrobial activity, through extraction with water:ethanol solvents (25:75, 50:50, and 75:25 (w/w)). It was analyzed the concentrated extracts antimicrobial activity against Escherichia coli, Klebsiella pneumoniae, Salmonella gallinarum, Staphylococcus aureus, and Staphylococcus epidermidis, and the quantification of the chlorogenic acids (3-CQA, 4-CQA, 5-CQA, 3,4-DQA, 3,5-DQA, and 4,5-DQA), p-coumaric acid, caffeine, and rutin. Only 75:25 extracts present positive antimicrobial effect; step 1 extracts present positive results for all the bacteria tested, step 2 presents positive results for Escherichia coli, and step 3 was positive for Escherichia coli and Staphylococcus epidermidis. The major phenolic compounds show that 50:50 and 25:50 extracts were more efficient to extract chlorogenic acids (p<0.05), while the processing step 2 may increase its concentration 3-CQA for 50:50 extract goes from 728.3 ± 31.2 µg/mL in step 1 to 2516.7 ± 29.7 µg/mL in step 2 and 2503.5 ± 40.3 µg/mL in step 3. Rutin and p-coumaric acid presents a similar behavior with the increasement of processing steps, however, only detected on 50:50 and 25:50 extracts of steps 2 and 3. Caffeine extraction ranged from 936.3 to 1170.2 µg/mL from all I. paraguariensis processing steps. The processing steps increasing was positive to increase phenolic compounds concentrations, while does not increase the antimicrobial activity. So, the antimicrobial activity should be positive due to other factors, as ethanol extract concentration or enzymes presents on the in natura leaves, not the major phenolic compounds.