The aim of this study was to determine the antibacterial activity of the essential oil, the extracts and pure metabolites of Origanum onites L. against plant pathogenic bacteria and potential use of the extracts against the diseases caused by some phytopathogenic bacteria in vitro and in vivo conditions. The essential oil and the extracts of O. onites, and its pure compounds isolated from the acetone extract were individually tested against a total of 14 phytopathogenic bacterial strains. The essential oil and hexane extract contained mainly carvacrol (70.50% and 80.50%), p-cymene (13.97% and 0.96%), thymol (2.19% and 7.53%, respectively). The oil showed potent antibacterial effect against all tested phytopathogenic bacteria, with inhibition zones of 22-40 mm. The hexane, chloroform and acetone extracts also inhibited the growth of the tested plant pathogenic bacteria exhibiting 12-24 mm inhibition zones, except Xanthomonas campestris pv. zinniae. MIC values for the oil and effective extracts were determined between 7.81 and 31.25 mu l/ml and 40 and 100 mg/ml, respectively. However, methanol extract did not show any antibacterial effects against the pathogenic bacteria. Seven compounds were isolated from acetone extract by column and thin layer chromatography and their chemical structures were characterized by UV, IR,H-1-NMR,C-13-NMR and 1D and 2D NMR spectroscopic methods as caryophyllene oxide, carvacrol, n-tetracosanol, beta-sitosterol, betulinic acid, ursolic acid, naringenin and aromadendrin. Among these compounds, carvacrol inhibited the growth of all the bacteria tested, whereas aromadendrin and naringenin inhibited only a few bacterial species. In the Petri plate assays, disease severity on the tomato and lettuce was reduced by the extracts as compared to control, whereas the high concentrations of the extracts showed a negative effect on seed germinations of the tomato and lettuce. The effects of the extracts on the germination, disease severity and seedling growth of tomato coated with Clavibacter michiganensis ssp. michiganensis, Xanthomonos axonopodies pv. vesicatoria and lettuce coated with Xanthomonas campestris pv. vitians were also studied on pots. The results showed that the extract applications significantly reduced the disease symptoms and they did not affect the germination and seedling growth of tomato and lettuce as compared to control. Furthermore, some applications increased the germination of tomato and lettuce. The current results showed that the extracts can be used in inhibiting some bacterial plant disease. (C) 2014 Elsevier B.V. All rights reserved.