Antimicrobial peptides (AMPs) are natural host-defense molecules essential to the innate immune responses of various organisms such as humans, animals, insects and plants. These peptides are being studied as templates for the next generation of potent antibiotics. More than 2500 AMPs have been discovered from natural sources and several other synthetic analogs have been developed. AMPs exhibit diverse sequences, secondary structures (α-helices, β-sheets), high cationicity and hydrophobicity. Their selectivity towards bacteria is mediated by electrostatic interactions between the cationic peptides and the anionic outer cell wall of bacteria. While it is clear that AMPs permeabilize bacterial membranes, details of the steps involved in antimicrobial attack are not well understood. This thesis presents a detailed characterization of the sequence of events during the action of AMPs on single, live E. coli bacteria using time-lapse fluorescence microscopy. Periplasmic GFP and SYTOX Green/Orange are used as reporter molecules for permeabilization of the outer and cytoplasmic membrane (OM and CM), respectively. Intervening white-light images enable monitoring of cell growth during AMP action. Cecropin A and LL-37, both natural AMPs, permeabilize the OM and CM sequentially within 30 min. Cell growth halts and cells shrink in length when the OM is disrupted. Septating cells are permeabilized earlier than non-septating cells. Curved membrane surfaces (endcaps and septa) are preferred over cylindrical surfaces. For the first time, the effects of two LUV-permeabilizing AMPs - *ARVA and *VAYR* were studied on single E. coli cells. Both peptides translocate across the OM without permeabilizing it to GFP. The CM is permeabilized first, evidenced by a change in the spatial distribution of GFP and rise of SYTOX Orange signal. After a short delay, GFP signal decays either rapidly (within ~30 seconds for *ARVA) or gradually (within ~15 min for *VAYR*). Implications on the stability of membrane disruptions caused by these peptides are discussed. Finally, this thesis also presents a characterization of post-permeabilization effects of AMPs on the spatial segregation of DNA and ribosomes in dividing bacteria.