Abstract: The molecules causing cytotoxicity to tumor cells having major research interest and their application to develop drug against tumor. The Coelomocytes constitute immune component of earthworm coelomic fluid have the potential to produce biomolecules which causes cytotoxicity to tumors. The present study comprises of culturing coleomocytes isolated from coelomic fluid of Eudrilus eugeniae in HBSS as a culture media and with suitable conditions and testing the activity of coelomic fluid ,culture supernatant sample by MTT assay on tumor cell lines lung cancer cells (A549)and colorectal tumor cell lines(HTC 116) which showed significant cell death compared to respective control cell. The percent cytotoxicity in coelomic fluid ,coelomocyte culture supernatant and dialyzed sample were in the order 85% ,95% and 62% respectively. The highest cyotoxicity showed by culture supernatant .Supernatant enhanced its utilization as the major source of cytotoxic agent against tumor. The results of MTT assay were validated by performing more sensitive assay against the selected cell line used in the present study. The coleomocyte culture supernatant was administered in the concentration range 1-2mg/ml to induce cytotoxicity.The results showed 90% cytotoxicity in A549 Cell lines with concentration of 2mg/ml.The colorectal tumor cell showed moderate compare to referred standard values of Indian pharmacopeia/FDA .The findings of the study provide scientific evidence to utilize coleomocyte culture supernatant as a source to develop anticancer drug for treatment of cancer.