Acorus calamus is a medicinal plant known for its multifaceted health benefits, especially against inflammation and infectious ailments. In the current work, we attempt to investigate the volatile constituents of A. calamus essential oil (ACEO) and, for the first time, to elucidate its antibacterial mechanism. Gas chromatography coupled to a mass spectrometer (GC-MS) was used to investigate phytochemical substances. The GC-MS analysis detected β-asarone (71.13 %), an aromatic anisole as main component, followed by α-asarone (12.07 %), β-calacorene (3.01 %), methyl isoeugenol (2.16 %). ACEO exhibited remarkable antibacterial activity, as demonstrated by significant inhibition zones against several bacteria that were tested. Gram-positive strains, Staphylococcus aureus and Bacillus subtilis, showed inhibition zones measuring 20.11 ± 0.28 mm and 18.06 ± 1.36 mm, respectively, while Gram-negative bacteria, Pseudomonas aeruginosa, and Escherichia coli, displayed slightly smaller zones of 15.58 ± 0.68 mm and 16.00 ± 0.04 mm. The results of ACEO were competitive with Tetracycline and Cefoperazone. Furthermore, ACEO demonstrated low MICs (ranging from 0.125 % to 1.0 %) and MBCs (ranging between 0.125 % and 2.0 %), with MBC/MIC ratios consistently below 4.0, confirming its bactericidal nature. Time-kill kinetics represented high lethality of ACEO at MIC levels against Staphylococcus aureus and Pseudomonas aeruginosa, resulting in a significant reduction in colony-forming units within 12-24 h. The antibacterial mechanisms of ACEO were investigated, demonstrating its potential to disrupt the integrity of the cell membrane and enhance membrane permeability. This disruption results in the release of genetic material (RNA and DNA) and proteins from the bacterial cell. Significant anti-biofilm activity further highlights the potential of the tested compounds in combating biofilm-associated infections. Scanning electron microscopy (SEM) images revealed significant inhibition of bacterial adhesion (First step of biofilm formation) on used surface as well as possible morphological changes in bacterial cells treated with ACEO.