The aim of this study was to use atomic force microscopy (AFM), an innovative type of microscopy, to investigate the different behaviours of erythromycin A (a 14-membered ring) and rokitamycin (a 16-membered ring) in disrupting the morphology of Streptococcus pyogenes with the M phenotype. AFM scanning and sensing of the topography of a sample makes it possible to obtain simultaneous high-resolution digital measurements of the x, y and z coordinates at any point on the bacteria surface. The images obtained before and 2, 4 and 6 h after incubation with erythromycin A (32 mg/L) and rokitamycin (2 mg/L) clearly show that not even high concentrations of erythromycin A interfere with the M phenotype of S. pyogenes, whereas rokitamycin has a progressive action that leads to the formation of abnormally large cells, the loosening of chain structure and the formation of clusters.