Moonlighting proteins are a group of proteins that, in addition to their canonical role, perform other, unrelated functions. Moonlighting activity is not caused by RNA splice variants, post-translational modifications or gene fusion. The number of detected moonlighting proteins is increasing rapidly and they can be found in eukaryotes, bacteria, archeae and even some viruses. Bacterial moonlighting proteins are the most abundant and they are extensively studied. It is common for originally cytosolic bacterial proteins to be exported to the cell surface in order to perform moonlighting activities. Once they are on the surface of the cell, they can participate in processes that contribute to the pathogenicity of bacteria. One such protein is the elongation factor Tu (EF-Tu) whose individual moonlighting roles are described in this paper. The canonical role of the EF-Tu protein is to catalyze the binding of aa-tRNA to the ribosome during the elongation phase of protein synthesis. In certain bacteria the EF-Tu protein can be found on the cell surface where it participates in bacterial adhesion, while exposure to the EF-Tu protein of some other bacteria can trigger the immune response in plants and animals. Since these kinds of moonlighting activities of EF-Tu protein are involved in bacterial infection, certain vaccines may be developed based on a recombinant EF-Tu protein. In addition to the role in pathogenicity, EF-Tu protein of some bacteria can also help maintain the shape of the cells through interactions with cytoskeletal proteins. Future research into moonlighting proteins will certainly contribute to understanding how genome and cell pathways function, but also help to develop new drugs and vaccines.