Pathogenic species of mycobacteria and corynebacteria possess a thick and impermeable cell wall that helps these organisms evade detection and treatment by denying drug and probe molecules access to their cytoplasm. A distinctive feature of this barrier is an additional hydrophobic outer membrane composed of mycolic acids, which are covalently bound to an arabinogalactan (AG) polysaccharide that provides a link to the cell surface peptidoglycan. Efforts to identify weaknesses in these outer defenses would benefit greatly from a more complete understanding of how its components are assembled and maintained throughout the life cycle of the organisms. This project aims to develop chemical tools that specifically target and exploit the extracellular mycolyltransferase enzymes Ag85A-C, which mediate the covalent attachment of mycolic acids to the cell wall AG. First, we will detail a strategy to covalently label the cell wall mycolyl-arabinogalactan (mAG) complex with fluorophores to enable the visualization of mAG biosynthesis in real time. Our approach involves the design and synthesis of mimics of the endogenous mycolyl donor trehalose monomycolate (TMM), which carry fluorophore-conjugated mycolic acids. These substrates will be added to living bacteria, where we will evaluate their recognition by Ag85 and the incorporation of their fluorescent cargo into the growing cell wall. We anticipate that this robust labeling strategy will allow the localization and dynamics of cell wall assembly to be studied in unprecedented detail. Finally, we describe an approach to develop an Ag85-triggered turn-on fluorescent probe designed to fluoresce specifically in the presence of low concentrations of mycobacteria. We envisage that such a chemical tool would facilitate the early detection of pathogenic mycobacteria.