Peptidoglycan is the main component of the bacterial cell wall. It is crucial for cell survival because it acts as a barrier between the internal contents of the cell and the outer environment; maintaining cellular integrity and shape. Despite its chemical structure being defined in great detail, its organisation in the cell at molecular level is still unknown. In this thesis, Atomic Force microscopy combined with quantitative image analysis were used to decipher the peptidoglycan molecular architecture in different bacterial strains and environments. First, in Chapter 3 a novel approach to study peptidoglycan using sacculi in liquid environment is presented and the structure of the external peptidoglycan for Staphylococcus aureus is explored in detail: the nascent areas are formed of concentric rings and the mature regions form a randomly oriented fibrous mesh. Then, in Chapter 4, the structure of the internal peptidoglycan surface of Staphylococcus aureus is directly imaged for the first time, consisting on a dense mesh with pores of ~ 6 nm. In contrast, the external mesh contains many pores larger than 30 nm in diameter. The septal plate architecture is presented as a complex structure formed by oriented rings on the external septal wall while disordered mesh on the internal surface. Next, in Chapter 5 a comprehensive comparison between different mutant strains is performed. Mutants lacking either hydrolysis or peptidoglycan synthesis enzymes result in significant differences in cell wall architecture. This is the first step towards molecular phenotypes. Another Gram-positive bacteria species, rod-shaped Bacillus subtilis is studied in Chapter 6 using the methodologies developed during previous chapters. The internal surface is highly ordered along the short circumferential axis. However, the septal plate presents a striking open mesh structure. Finally, the addition of different antibiotics causes critical big holes that perforate through the cell wall not observed on healthy Staphylococcus aureus cells. This answers the 80 years old question of how antibiotics work, which is key to defeat the antimicrobial resistance crisis.