The search for new antimicrobials is essential to address the worldwide issue of antibiotic resistance. The present work aimed at assessing the antimicrobial activity of Aesculus hippocastanum L. (horse chestnut) bark against bacteria involved in urinary tract infections (UTIs). Bioactive compounds were extracted from A. hippocastanum bark using water and ethanol as solvents. The extracts were tested against 10 clinical uropathogenic strains including five Gram-positive and five Gram-negative bacteria. Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 25922 were used as reference bacteria. The susceptibility to antibiotics was assessed using the Kirby Bauer disc diffusion method and the antibacterial activity of the extracts was evaluated using the well diffusion method. The Minimum inhibitory concentration (MIC) and the minimum bactericidal concentrations (MBC) were asseded by the microdilution method. A. hippocastanum bark possessed a dry matter content of 65.73%. The aqueous extract (AE) and ethanolic extract (EE) showed a volume yield of 77.77% and 74.07% (v/v), and a mass yields of 13.4% and 24.3% (w/w) respectively. All the bacteria were susceptible to amoxiclav, imipenem and ceftriaxone but the clinical strains were resistant to at least one antibiotic. Kocuria rizophilia 1542 and Corynebacterium spp 1638 were the most resistant bacteria both with multidrug resistance index of 0.45. Except AE on Proteus Mirabilis 1543 and Enterococcus faecalis 5960 (0 mm), both AE and EE were active against all the microorganisms tested with inhibition diameters (mm) which ranged from 5.5-10.0 for AE and 8.0-14.5 for EE. The MICs of EEs varied from 1-4 mg/mL while those of AEs varied from 4-16 mg/mL. The ethanolic extracts (EE) were overall more active than the aqueous ones. The A. hippocastanum bark extracts had overall weak antibacterial activity (MIC ≥0.625 mg/mL) and bacteriostatic potential (MBC/MIC ≥16) on both Gram-positive and Gram-negative bacteria. © 2022 The Author(s). Published by IMR Press.