Bacillus clausii, a gram positive, spore forming bacteria is widely used a probiotic in preventing and treating various gastrointestinal disorders, are known to colonize in host systems. But Staphylococcus aureus, though gram positive and show ability to colonize in host systems, is pathogenic in nature. In order to explore the nature of interaction of these bacteria to their hosts, a molecular level study was proposed primarily to isolate and characterize surface layer and secretory proteins. The surface layer proteins were extracted through lithium chloride treatment followed by their isolation and purification by fibronectin and gelatin affinity chromatography. SDS PAGE analysis showed few gelatin binding proteins from Bacillus clausii. Similar observations were made from surface proteins isolated off Bacillus clausii and Staphylococcus aureus through fibronectin affinity chromatography.