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The continual emergence of pathogen resistance is a recurring challenge and pushes for the development of antimicrobial compounds. Here, we investigated compounds from quaking aspen trees ( Populus tremuloides ) as potential antimicrobial agents. Several extractions using different solvents were realized, and corresponding antimicrobial activity was tested against eight microorganisms. Results revealed that polar extraction solvents including water, ethanol and methanol gave the best extraction yields (>15.07%). Minimal inhibition concentration (MIC) and minimal bactericidal/fungicidal concentration (MBC/MFC) demonstrated that water extracts had the best antimicrobial activity by a weak to moderate inhibition of growth of all eight tested microorganisms in addition to having a bactericidal effect on three of them. The quaking aspen methanol extract also displayed antimicrobial activity but to a lower level than the water extract. Ultra-performance liquid chromatography quadrupole time-of flight mass spectrometry (UPLC-QTOF-MS) analysis led to the identification of 92 compounds, mainly polyphenols in both extracts, with 22 molecules previously known for their antimicrobial properties. According to the relative abundance, 4-hydroxybenzaldehyde (5.44% in methanol extract) and kaempferol (5.03% in water extract) were the most abundant antimicrobial compounds. Among antimicrobial molecules identified, nine were from the flavonoid family. The results of our study demonstrate the interest of using quaking aspen as source of antimicrobial compounds.