Hirsutella thompsonii # 966 cultured in malt extract broth for 1 month without air flow was used to analyze chemical components by extraction with ETOAC. The components were separated with a Sephadex LH-20 column and analyzed on a Bruker DRX 400 NMR spectrometer. No new compounds appeared in the purification process which showed only a group of ordinary components : peptide, glycerol derivative, ergosterol and diketopiperazines A and B,.so, the extraction process was terminated. The crude broth ( freeze dried sample ) and the crude extract from broth ( after evaporation ) were used to investigate the cytotoxicity against insect and mammalian cell lines by dissolving in dimethyl sulfoxide ( DMSO ). Two insect cell lines, Sf9 ( pupal ovarian tissue of Spodoptera frugiperda Smith = ECACC No. 89070101 ) and Clone C6/36 ( larvae tissue of Aedes albopictus Skuse = ECACC No. 89051705 ) plus one mammalian cell line, clone BHK ( 21 ) clone 13 ( Hamster Syrian kidney = ECACC No. 85011433 ) were used in this study. A bioassay revealed that the crude broth sample was weakly toxic to all cell lines in the tested range of 7.8 – 1,000 ?g/ml while the crude extract was non-toxic to all cell lines tested in the range of 0.78 – 100 ?g/ml.