Aspergillus fumigtus produced a protenaceous hemolysin, when incubated on sheeps blood agar, from 41 isolates thirty isolates (73.1%) has able to produce hemolysin. Hemolysin purified from tryptic soy broth using ion exchang and gel filtaration and found to have amolecular weight of approximately 74.52 KD a. In this study, hemolysin was used in an experimental model to study its Cytotoxic activity by evaluating effect on REF cell line (Rat embryonic fibroblast), on exposure time of  24  hrs at three different concentrations triplicate of each concentration  were used, Cytotoxicity of the purified compounds are  active against REF cell line under study and a toxic effect was clear with a significant difference at the level of probability (p< 0.05) and this effect was increase gradually with the increase of hemolysin concentration.