Abstract: Background and Objective: Oil extracts from the seeds of Rhus semialata were screened and used for antimicrobial activity against bacterial strains-Staphylococcus aureus, Escherichia coli and Klebsiella pneumoneae and fungal strains-Aspergillus niger and Penicillium sp. Current study was aimed to scientifically validate and document the antimicrobial properties of R. semialata seeds. Materials and Methods: The plant material extracts were carried out with non polar and highly polar solvents for oil extraction using petroleum ether, chloroform and methanol for phytochemical, antimicrobial and Gas Chromatography and Mass Spectroscopy (GCMS) studies. Results: Extracts contains a wide range of medicinally active components includes organic acids, alkaloids, phenols, flavonoids, tannins, fixed oils, fats and terpenoids by phytochemical analysis. GCMS studies showed more than 31 compounds among them the major compounds observed was tridecane (57%), oxalic acid, isobutyl nonyl ester (43%), nonane and decane (57%). Antimicrobial activity of R. semialata seed extract exhibited maximum zone of inhibition (25 mm) at 100 μL concentration of chloroform. Conclusion: The traditional use of the extracts in infections and inflammatory conditions is rationalized based on the active principles present in the oils and it has been formulated for use in many infections of bacterial and fungal pathogens.