The composition of essential oils obtained from leaves of Acorus calamus growing wild in 19 different locations was analysed by gas chromatography and gas chromatography–mass spectrometry. In total, 84 constituents have been identified, representing at least 86% of the total essential oil. The essential oils were dominated by the presence of phenolic compounds: (Z)-asarone (15.7–25.5%) and (Z)-methyl isoeugenol (2.0–4.9%). Other identified major components were (E)-caryophyllene, α-humulene, germacrene, linalool, camphor and isoborneol. All oils were found to have a similar composition. However, a hierarchical cluster analysis of the quantitative data revealed significant differences between some accessions. Minimal inhibitory concentrations (MIC) of four selected leaf essential oils were determined by a screening test with the broth dilution method. Of the 17 species of fungi, bacteria and yeasts tested, Mycobacterium ssp. and Bacillus subtilis were most susceptible to essential oils. Oils displayed low anti-fungal activity and their inhibitory activity was lower than the standard antibiotics. Oils differed in their activities against the various microorganisms tested.