In the present study, chemical composition and the antibacterial mechanism of ambrette seed oil are investigated. Chemical composition of the oil was analysed by gas chromatography-mass spectrometry (GC-MS). Thirty-five compounds were identified and the major compounds were found to be farnesol acetate (51.45%) and ambrettolide (12.96%). The antibacterial activity was performed by well diffusion assay and the mechanisms were studied by measuring the alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and protein leakage assays. The antibacterial effect of the ambrette seed oil showed inhibitory effect against Bacillus subtilis, Staphylococcus aureus and Enterococcus faecalis. The LDH activity was high in all tested bacteria compared with control, whereas the ALP and protein concentrations were also increased in E. faecalis. Molecular docking revealed the ligands farnesol acetate and ambrettolide had satisfactory binding energy towards the beta lactamase TEM-72 and dihydrofolate reductase (DHFR) protein. Due to its better antibacterial properties, the ambrette seed oil could be used as a source of antibacterial agents.