Pimenta dioica (Linn.) Merill., belongs to the family Myrtaceous, has been used as a spice and is well known for its culinary as well as medicinal values. The aim of the current study was to assess the in vitro antimicrobial, anthelmintic activity and phytochemical composition of the essential oil extracted from Pimenta dioica leaves. The extraction of essential oil was performed with the Clevenger apparatus, by hydro-distillation. Pimenta dioica essential oil yield was 1.03%. The GC-MS analysis of this Pimenta dioica essential oil showed that eugenol (74.72%) was the predominant component. The essential oil showed potent anthelmintic activity assay when analysed by the adult worm’s motility assay. It has shown a comparable paralysis and disintegration profile of the model organism to that of the standard drug albendazole. The essential oil was also found to be effective against common human pathogenic bacteria, including Staphylococcus aureus, Klebsiella, Escherichia coli, Chromobacterium violaceum and Proteus vulgaris. It also showed moderate antifungal activity against Candida albicans and Aspergillus niger. These results proved that Pimenta dioica essential oil could be used as a source of the lead molecule for developing novel anthelmintic or antimicrobial agents.