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Zataria multiflora Boiss. belonging to the family Lamiaceae is an important folkloric medicinal plant in Fars province, Southwestern Iran. In this study, essential oil constituents, phenolic content, antioxidant and antimicrobial activities from the aerial parts of Z. multiflora collected from different natural habitats of Fars province were investigated. The essential oil of all samples was extracted by hydro-distillation using Clevenger type apparatus and analyzed using GC and GC-MS. The main chemical constituents were, thymol (34.41 - 54.35 %), p-cymene (9.49 - 19.85 %), γ-terpinene (7.34 - 16.70 %), carvacrol (5.35 - 15.34 %) and α-pinene (1.63 - 5.25 %). The total phenolic content and the antioxidant activity of methanolic extracts were determined with the Folin-Ciocalteau reagent and by the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, respectively. Total phenols varied from 234.66 to 302.28 mg gallic acid equivalents/g dry weight, and IC50 values in the radical scavenging assay ranged from 348.63 to 453.76 mg/mL. The antimicrobial activity of the essential oils was individually evaluated against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Candida albicans using disc diffusion and serial dilution methods. The oils obtained from different geographical habitat showed high activity against four medically important pathogens. The results support the traditional usage and also possible use of Z. multiflora essential oil and extracts in the food, pharmaceutical and cosmetic industries. [ABSTRACT FROM AUTHOR]