Background: Zataria multiflora Boiss. is a traditional and popular spice in Iran. The effects of 3 ecotypes (ECTPs) of Z. multiflora essential oils (EOs) against most common causes of food-borne and nosocomial infections were evaluated. Materials and Methods: The antimicrobial activities of the EOs were examined by broth microdilution method as recommended by the Clinical and Laboratory Standards Institute (CLSI). The chemical compositions of the EOs from 3 ECTPs of Z. multiflora have been analyzed by gas chromatography-mass spectrometry. Results: Analysis of the EOs indicated that 3 chemotypes were present in Z. multiflora, including carvacrol, thymol-carvacrol, and linalool, whereas previous studies have only found carvacrol and thymol. Inhibition studies showed that the tested EOs entirely inhibited the growth of yeasts at concentrations of less than 1 μL/mL. Moreover, the oils exhibited significant bacteriostatic and bactericidal activities against Gram-positive and Gram-negative bacteria at concentrations ranging from 0.12 to 8 μL/mL. Conclusion: These results suggest that the EOs from Z. multiflora should be investigated further for possible use in antimicrobial products and food preservatives.