Bacterial secretome is a comprehensive catalog of bacterial proteins that are released or secreted outside the cells. They offer a number of factors that possess several significant roles in virulence as well as cell to cell communication and hence play a core role in bacterial pathogenesis. Sometimes these proteins are bounded with membranes giving them the shape of vesicles called extracellular vesicles (EVs) or outer membrane vesicles (OMVs). Bacteria secrete these proteins via Sec and Tat pathways into the periplasm. Secreted proteins have found to be important as diagnostic markers as well as antigenic factors for the development of an effective candidate vaccine. Recently, the research in the field of secretomics is growing up and getting more interesting due to their direct involvement in the pathogenesis of the microorganisms leading to the infection. Many pathogenic bacteria have been studied for their secretome and the results illustrated novel antigens. This review highlights the secretome studies of different pathogenic bacteria in humans and animals, general secretion mechanisms, different approaches and challenges in the secretome of Mycoplasma sp.