The enormous increase in our population has necessitated more and more food production through alternate resources such as mushroom. Pleurotus mushrooms are the second most important mushroom in its production in the world. The present investigation deals with Gas Chromatography [GC]-Mass Spectrometry [MS] analysis of petroleum ether extract of dried fruiting bodies of Pleurotus eous and its evaluation of antibacterial activity. Five compounds were identified by GC-MS and they are fatty acids esters such as Cyclopentanetridecanoic acid, methyl ester (25%); Tartronic acid, (p-ethoxyphenyl), diethyl ester (16.3%); 7, 10-Octadecadenoic acid, methyl ester (40.3%); Heptadecanoic acid, 16-methyl, methyl ester (13.5%) and 9-Octadecenoic acid [Z]-, 2-hydroxyl-1-[hydroxymethyl] ethyl ester (4.9%). This is the first report on the composition of the fatty acids of these fruiting bodies. Petroleum ether, ethyl acetate, methanol and aqueous extracts of P.eous were obtained by hot continuous Soxhlet extraction and were screened for its antibacterial property against Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae. Among all the crude extracts tested, only petroleum ether extract showed strong antibacterial activity by inhibiting the growth of both gram positive and gram negative bacterial isolates. The minimal inhibitory concentration (MIC) values of the petroleum ether extract against S. aureus is (4.4μg/ml), B.subtilis (3.1μg/ml), B.cereus (4.2μg/ml), P.aeruginosa (8.8μg/ml), E.coli (3.1μg/ml) and K. pneumoniae (4.4μg/ml). Hence, based on these observations it can be clearly concluded that petroleum ether extract is found to have antimicrobial activity. The GC-MS studies on this extract further confirms that this activity is due to the presence of fatty acid esters that are identified in the present study. The investigation therefore supports the traditional uses of the mushroom in the treatment of infectious diseases.