Bacterial resistance to conventional antibiotics is of major concern. Antimicrobial peptides (AMPs) are considered excellent alternatives. Among them, D-cateslytin (D-Ctl, derivative of a host defense peptide) has shown high efficiency against a broad spectrum of bacteria. The first target of AMPs is the outer membrane of the bacterium. However, the role of bacterial cell-wall structures on D-Ctl’s mechanism of action has not yet been understood. In this study, we investigated the activity of D-Ctl on two isogenic strains of E. coli: one is devoid of any parietal structures; the other constitutively overexpresses only type 1 fimbriae. We studied the damage caused by D-Ctl at several initial concentrations of bacteria and D-Ctl, and exposure times to D-Ctl were examined using a combination of epifluorescence microscopy, atomic force microscopy (AFM), and Fourier transform infrared spectroscopy in attenuated total reflectance mode (ATR-FTIR). The analysis of nanomechanical and spectrochemical properties related to the antibacterial mechanism showed a concentration dependent activity. Whereas the membrane permeabilization was evidenced for all concentrations of D-Ctl and both mutants, no pore formation was observed. The bacterial stiffness is modified dramatically concomitantly to major membrane damage and changes in the spectral fingerprints of the bacteria. In the case of the occurrence of type 1 fimbriae only, an intracellular activity was additionally detected. Our results evidenced that D-Ctl activity is highly impacted by the cell-wall external structures and surface properties of the bacteria.