Abstract: We examined the effect of plant part, cultivating location, harvest season and plant age on the oil yield, chemical composition and antibacterial activity of essential oils from rosemary (Rosmarinus officinalis). We analyzed essential oils by gas chromatography-mass spectrometry and compared the antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Proteus vulgaris. All oil samples showed antibacterial ability against the 4 strains. For Guizhou rosemary, the major compounds of leaf oils were α-pinene (31.88%–39.09%), 1,8-cineole (8.82%–10.97%) and camphor (7.63%–11.59%), whereas those of stem oils were borneol (26.62%–39.05%), camphor (11.41%–15.27%) and α-pinene (7.23%–17.80%). Antibacterial activity was greater for oils from stems than leaves. For oils from Beijing rosemary, the major compounds of leaf oils were camphor (27.92%) and L-β-pinene (25.28%), and the oils showed higher antibacterial activity than those from Guizhou, whereas oils from Beijing plant stems were similar to those from Guizhou plant stems. Oil yields were higher and antibacterial activity stronger with harvesting in June than September and December. In terms of plant age, oil yield, chemical composition and antibacterial activity showed slight differences. Rosemary could be cultivated in parks of Beijing as an ornamental plant and in mountain areas of Guizhou for economic use. Rosemary might be harvested twice in summer and winter, with autumn the second vegetative growing period. Moreover, both leaf and stem parts of rosemary can be used for at least 10 years with little degradation. Our study provides a theoretical basis for maximizing the use of rosemary.