BACKGROUND: The rise in multidrug-resistant bacteria has intensified the search for new antibiotics, drawing attention to essential oils (EOs) for their antimicrobial properties. For this reason, this study focuses on the antimicrobial action of the EO obtained from Tagetes minuta and its impact on bacterial membranes. METHODS: The EO was chemically characterized by chromatography-mass spectrometry, and its antimicrobial activity and its effects on surface and bacterial membrane were assessed by using Zeta potential, membrane transition temperature (Tm) determination; and fluorescence spectroscopy with Laurdan and Di-8 ANEPPS. RESULTS: Twenty-seven compounds could be identified, with (E)-Tagetone, (Z)-Ocimenone, and β-pinene as the most abundant. Afterward, the EO was tested against Escherichia coli (MIC and MBC = 17 mg/mL) and Staphylococcus aureus (MIC = 8.5 mg/mL; MBC > 17 mg/mL), showing antimicrobial action in both bacteria, being more effective against E. coli. Mechanistic studies revealed that the EO interacts with bacterial membranes, increasing the Zeta potential by more than 9 mV and enhancing membrane permeability up to 90%. These effects were further confirmed using model lipid membranes, where the EO induced significant changes in membrane properties, including a reduction in dipole potential and transition temperature, suggesting that some EO components could be inserted into the lipid bilayer, disrupting membrane integrity. CONCLUSIONS: The EO from T. minuta demonstrates efficient antimicrobial activity by compromising bacterial membrane structure, highlighting its potential as a natural antimicrobial agent.