Interest in nanotopographies as potential antibacterial surface coating have gained significant attraction due to its unique antimicrobial mechanism [1]. It was reported in several studies that nanotopographies can induce mechanical rupture of the cell membrane upon contact which could provide an effective approach to prevent infections on medical implants and devices whilst reducing our dependent on antibiotics. Despite several theories proposed on the mechanistic behaviour of the nanotopographies, no consensus has been reached on the precise mechanism of bacteria-nanotopography interaction [2]. To assess the antibacterial mechanism of synthetic nanotopographies, it is crucial to analyse the bacteria-nanotopography adhesion interface. Here, we utilise focused ion beam (FIB) milling combined with scanning electron microscopy (SEM) to generate three-dimensional reconstructions of whole bacteria (Staphylococcus aureus and Escherichia coli), enabling bacteria-nanotopography interactions to be resolved with nanometer resolution. 3D morphometric analysis was used to quantify the intrinsic contact area between each nanostructure and the bacterial envelope, the first time this approach has been used to study bacteria-nanotopography interactions. In the case of significant changes to the cell morphology was observed, the damage is most likely due to the penetration of the nanostructures which was seen in both E. coli (Figure 1) and S. aureus cells. We also found evidence of physical deformation and cell impedance that induces stress response from the bacteria. The FIB-SEM and morphometric analysis allows the quantification of the proportion of nanostructures penetration per bacteria, the effective contact surface area for each interactions and changes in cell dimension. These metrics provide important parameters to derive accurate mechanistic model of interaction between bacteria and nanotopography.