(1) Antimicrobial peptides (AMPs) are a promising alternative to conventional antibiotics. Among AMPs, the disulfide-rich beta-defensin AvBD103b, whose antibacterial activities are not inhibited by salts contrary to most other -defensins, is particularly appealing. Information about the mechanisms of action is mandatory for the development and approval of new drugs. However, data for non-membrane-disruptive AMPs such as beta-defensins are scarce, thus they still remain poorly understood. (2)We used single-cell fluorescence imaging to monitor the effects of a beta-defensin (namely AvBD103b) in real time, on living E. coli, and at the physiological concentration of salts. (3)We obtained key parameters to dissect the mechanism of action. The cascade of events, inferred from our precise timing of membrane permeabilization effects, associated with the timing of bacterial growth arrest, differs significantly from the other antimicrobial compounds that we previously studied in the same physiological conditions. Moreover, the AvBD103b mechanism does not involve significant stereo-selective interaction with any chiral partner, at any step of the process. (4) The results are consistent with the suggestion that after penetrating the outer membrane and the cytoplasmic membrane, AvBD103b interacts non-specifically with a variety of polyanionic targets, leading indirectly to cell death.