Present work is directed toward the isolation and identification of biological active (antimicrobial) compound from metabolite extracted from endophytic fungus Emericella qaudrilineata(RS-5) (Accession number KC662361). Crude extracted from endophytic fungus Emericella qaudrilineata possessing antibacterial activity was subjected to bioassay guided fractionation in which toluene fraction exhibited maximum antibacterial activity against Staphylococcus aureus (gram positive) and Aeromonas hydrophilla (gram negative) respectively. Sequentially compounds fractionated from ethyl acetate and hexane also shown feeble antibacterial activity. This reveals that toluene fraction contain active principle of antimicrobial interest. In continuation to above experiment, major active fractions were analyzed with the help of gas chromatography mass spectroscopy (GCMS). GCMS analysis of toluene fraction showed benzyl benzoate (27.3%), benzaldehyde dimethyl acetal (15.04%) and benzoic acid (17%), as the dominant compound present in fraction of toluene. In context to above details separation of compound was performed by silica gel column chromatography. Separated band showed its presence at 0.6 RF value in silica gel (TLC) plates. Separated compound also exhibited remarkable antibacterial activity. The optimized flow rate of column was 20 ml/min in ratio of hexane 20:80 toulene mobile phase. Prediction of C 13 NMR, H 1 NMR and FTIR data supported the dominating compound of GCMS profile of toulene fraction, and identified it as Benzyl benzoate. Medically benzyl benzoate is an active ingredient of Ascabiol that is generally used to treat scabies various skin diseases.