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Antimicrobial resistance is a global health issue, in which microorganisms develop resistance to antimicrobial drugs, making infections more difficult to treat. This threatens the effectiveness of standard medical treatments and necessitates the urgent development of new strategies to combat resistant microbes. Studies have increasingly explored natural sources of new antimicrobial agents that harness the rich diversity of compounds found in plant species. This pursuit holds promise for the discovery of novel treatments for combating antimicrobial resistance. In this context, the chemical composition, antibacterial, and antibiofilm activities of the essential oil from Croton urticifolius Lam. leaves (CuEO) were evaluated. CuEO was extracted via hydrodistillation, and its chemical constituents were identified via gas chromatography-mass spectrometry (GC/MS). The antibacterial activity of CuEO was evaluated in a 96-well plate via the microdilution method, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were determined. The effect of CuEO on biofilm formation was assessed by quantifying the biomass using crystal violet staining and viable cell counting. In addition, alterations in the cellular morphology of biofilms treated with CuEO were examined using scanning electron microscopy (SEM) and laser confocal microscopy. GC/MS analysis identified 26 compounds, with elemicine (39.72%); eucalyptol (19.03%), E-caryophyllene (5.36%), and methyleugenol (4.12%) as the major compounds. In terms of antibacterial activity, CuEO showed bacteriostatic effects against Staphylococcus aureus ATCC 700698, S. aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, and Escherichia coli ATCC 11303, and bactericidal activity against S. aureus ATCC 700698. In addition, CuEO significantly inhibited bacterial biofilm formation. Microscopic analysis showed that CuEO damaged the bacterial membrane by leaching out the cytoplasmic content. Therefore, the results of this study show that the essential oil of C. urticifolius may be a promising natural alternative for preventing infections caused by bacterial biofilms. This study is the first to report the antibiofilm activity of C. urticifolius essential oil., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)