The composition of the essential oils obtained by hydrodistillation from different parts of Bupleurum rotundifolium L. including roots, flowers and fruits were investigated by GC and GC/MS systems, simultaneously. The antibacterial activity of the oils was assessed with micro-dilution assay. The results showed large variations in the composition of the oils. The main components of the plant were undecane (26.4%) and tridecane (12.3%) in the roots, hexadecanoic acid (12.2%) in the flowers and alpha-pinene (11.0%) in the fruits. The essential oils of B. rotundifolium obtained from flowers and fruits used in the study did not have any effect against bacteria. MIC values for the bacterial strains tested, which were sensitive to the essential oil of roots of B. rotundifolium, were in the ratio of 2 mg/mL. This investigation showed that the antibacterial activity of B. rotundifolium was attributed to the essential oil of roots.