Roots of wild growing Angelica archangelica L. from Mt. Ozren (Serbia) were subjected to hydrodistillation and GC-MS analysis. The roots contained 0.10% of essential oil with a-pinene (29.7%), 8-3-carene (14.2%), and a mixture of P-phellandrene and limonene (13.2%) as main compounds. The modified resazurin microtiter-plate assay was used to evaluate the antibacterial activity of the essential oil against Staphylococcus aureus and Escherichia coli. The minimum inhibitory concentration (MIC) values were 14.2 μL/mL for S. aureus and 28.4 μL/mL for E. coli, while the minimum bactericidal concentrations (MBC) were 56.8 μL/mL and 113.6 μL/mL, respectively. According to the obtained results, the angelica root essential oil can be applied as a natural preservative in food and as a natural antibiotic for the treatment of several infectious diseases caused by these two bacteria.