The coelomic fluid (CF) is the body fluid present in the space between the body wall and gut in the earthworms. The earthworm’s coelomic fluid contains various molecules and exhibits important biological properties such as antimicrobial substances, hemagglutination and anticoagulant agents. The coelomic fluid of earthworm possesses primary function that has the ability to destroy the membranes of foreign cells. It is a mechanism that leads to cell death by the release of cytosol and attributes coelomocytes which secretes humoral effectors into the coelomic fluid. In this work, bacterial cultures namely Pseudomonas aeruginosa and Bacillus subtilis were injected into the earthworm body cavity. Then the coelomic fluid extract from the bacteria injected earthworms were collected out. The extracted coelomic fluids were partially purified and precipitated using ammonium sulphate and analyzed to biological activities such as antibacterial activity, proteases activity and heamolytic activity. Then the active compounds present in the coelomic fluids were analysed to FTIR, HPLC and LCMS. This concludes that the microbes introduced into the earthworm body containing coelomic fluids have lysenin protein that inhibits antibacterial, protease and haemolytic activity. Hence the induction of bacterial cultures Pseudomonas aeruginosa and Bacillus subtilis into the earthworm’s body cavity leads to the secretion of lysenin protein in the coelomic fluid of earthworms. Thus the presence of proteins presence in the coelomic fluid can be used for pharmaceutical purposes.