The present study was conducted to investigate the chemical composition of Ailanthus
altissima (Mill.) Swingle methanolic leaf extracts from geographically distinct regions and to assess
their antimicrobial properties along with their ability to induce oxidative stress. The HPLC-DAD
analysis revealed the presence of phenolic acids and flavonoids including chlorogenic acid, gallic acid,
synapic acid, p-coumaric acid, apigenin, hyperoside, isoamnétine-3-O-beta-D-glucotrioside, quercetin,
and isoquercetin in various amounts depending on the origin of tested extracts. The assessment
of antibacterial activity showed the effectiveness of the A. altissima extracts particularly against
Gram-positive bacteria, with inhibition zone diameters reaching 14  1 mm and minimum inhibitory
concentrations ranging from 4 to 72.2 mg/mL. These bioactive substances also exhibited strong
antibiofilm activity with an eradication percentage reaching 67.07%. Furthermore, they increased
ROS production to levels two to five times higher than the control group, altered the membrane
integrity and caused lipid peroxidation with MDA production exceeding 2.5 mol/mg protein in the
Gram-positive and Gram-negative strains. A decrease in the levels of the antioxidant enzymes SOD
and CAT was also observed, indicating an impairment of the bacterial response to the oxidative stress
caused by the tested extracts. These findings highlight the antibacterial properties of A. altissima leaf
extracts depending on their origins and promote their exploitation and application in the agro-food
and pharmaceutical sectors.