The fruit of Ehretia acuminata R. Br. is used in eating by some tribal people and is a good source of antioxidants found in Asia, Australia, Africa, and North America. Our study aims to investigate the extraction of essential oil from the fruit of E. acuminata, the evaluation of physicochemical parameters, chemical composition, and biological activities of essential oil. The essential oil was extracted by hydrodistillation method, physicochemical parameters like yield, colour, odour & refractive index, acid value, iodine value, saponification value, ester value & peroxide value, assessing the composition of the essential oil were determined by gas chromatography/mass spectrometry. The evaluation of different biological activities like antioxidant, antidiabetic, anti-inflammatory, antihemolytic, and photoprotective activities have been done by the spectrophotometric method and antimicrobial by zone inhibition method. The results obtained from GC/MS analysis proved the presence of 30 components, in which 29 compounds were identified, with the highest content of 2,4-Decadienal (37.54%), (E, E)-2,4-Decadienal (20.31%), 2-(Prop-2-benzyloxy) tetradecane (10.00 %), Diethyl Phthalate (5.28%). The chemical composition and biological activity of the investigated essential oil indicate a significant phytomedical potential of the essential oil of E. acuminata fruit.