Abstract: The shape and integrity of bacteria are determined by cell wall peptidoglycan, a single macromolecule that surrounds the cell. The synthesis of peptidoglycan is also the site of action of important antibiotics such as penicillin and vancomycin. Thus, bacterial viability, growth and division are dependent on the architecture and dynamics of this essential polymer. We use a range of high-resolution microscopy approaches, coupled with biochemical, genetic and physiological approaches to reveal peptidoglycan architecture, and its dynamics, using the major human pathogen Staphylococcus aureus as our primary organism. Atomic force microscopy has demonstrated a complex, nanoscale peptidoglycan architecture in diverse species, which meets the challenges of maintaining viability and growth within their environmental niches, by exploiting the bioengineering versatility of the polymer. The application of super-resolution fluorescence microscopy, coupled with new chemical probes has begun to reveal how the polymer is synthesized and hydrolysed during growth and division. We have also used these approaches to determine the mode of action of antibiotics and molecular mechanisms of antimicrobial resistance.