The effects of native and semisynthetic beta-lactam on the surface of E. coli cells were investigated using tapping mode atomic force microscopy (AFM). These studies were done on both single cell and cell colonies. This allowed us to achieve a complete understanding of the effects, since previous related AFM analysis was only performed on single cells. Our results indicated that although amoxycillin and penicillin could both induce nanoporous damage to the envelope of E. coli, the distribution of the pores was different: those induced by penicillin were randomly distributed on the cell surface, while those induced by amoxycillin were far more numerous and mainly on the two ends of the cell. These findings could explain why the effect of amoxycillin is stronger than that of native penicillin. Ofloxacin was used as a control due to its inactive membrane, and no cell wall damages were observed. Our results demonstrate that AFM is a useful tool in discerning and verifying antibiotic mechanisms and can be helpful to explain the relationship between chemical structure and the function of antibiotics.