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The principal objective of the present study is to evaluate both the chemical composition and antibacterial effectiveness of essential oil of Clinopodium nepeta subs ascendens (CNEO) against three Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus, and Bacillus subtilis) and three Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae). Additionally, this research aims to explore potential synergies between CNEO and established antibiotics. The phytochemical composition of CNEO was thoroughly examined via gas chromatography mass spectrometry (GC-MS), revealing neoisomenthol (37.89%), pulegone (20.11%), and dihydrocarvone (19.01%) as the primary bioactive molecules. CNEO exhibited substantial antibacterial activity, with minimum inhibitory concentration (MIC) values ranging from 0.10 against E. coli to 1 mg/mL against K. pneumoniae. This promising antimicrobial activity, especially noteworthy against antibiotic-resistant bacteria, prompted an evaluation of CNEO's synergistic potential with antibiotics using the Fractional Inhibitory Concentration Index (FICI). The FICI analysis, especially with ampicillin against P. aeruginosa, uncovered an optimal synergistic interaction at a FICI of 0.31. Notably, observed reductions in MIC values for CNEO and antibiotics, ranging from 2-7 folds, highlight the heightened efficacy of these combinations. These findings hold significance for developing new approaches to address the increasing challenges of bacterial infections resistant to conventional treatments. [ABSTRACT FROM AUTHOR]