Four essential oils from the leaf (P23) and rhizomes (P19, P22, P24) of Acorus calamus L., collected from various parts of Nepal, were obtained by hydrodistillation and analyzed by GC-MS. From a total of 61 peaks, 57 compounds were identified among the four essential oils accounting for 94.3%, 96.2%, 97.6%, and 94.1% of the oils, respectively. All of the essential oils were dominated by (Z)-asarone (78.1%-86.9%). The essential oils also contained (E)-asarone (1.9%-9.9%) and small amounts of gamma-asarone (2.0-2.3%), (Z)-methyl isoeugenol (1.5-2.0%), and linalool (0.2-4.3%). Allelopathic testing of the rhizome oil showed inhibition of seed germination of Lactuca sativa and Lolium perenne with IC50 values of 450 and 737 microg/mL, respectively. The rhizome essential oil demonstrated stronger seedling growth inhibition of L. perenne than of L. sativa, however. The rhizome oil also showed notable brine shrimp lethality (LC50 = 9.48 microg/mL), cytotoxic activity (92.2% kill on MCF-7 cells at 100 microg/mL), and antifungal activity against Aspergillus niger (MIC = 19.5 microg/mL).