The virulence of Staphylococcus aureus, a multi-drug resistant pathogen, notably is determined by the expression of the phenol soluble modulins α3 (PSMα3) peptides, in a position to self-assemble into amyloid-like cross-α fibrils. Regardless of outstanding advances evidencing the essential, but inadequate, function of fibrils in PSMα3 cytotoxic actions in direction of host cells, the connection between its molecular buildings, meeting propensities, and modes of motion stays an open intriguing downside. On this research, combining atomic pressure microscopy (AFM) imaging and infrared spectroscopy, we first demonstrated in vitro that the cost supplied by the N-terminal capping of PSMα3 alters its interactions with mannequin membranes of managed lipid composition with out compromising its fibrillation kinetics or morphology. N-formylation finally dictates PSMα3-membrane binding by way of electrostatic interactions with the lipid head teams. Moreover, PSMα3 insertion throughout the lipid bilayer is favoured by hydrophobic interactions with the lipid acyl chains solely within the fluid part of membranes and never within the gel-like ordered domains. Strikingly, our real-time AFM imaging emphasizes how intermediate protofibrillar entities, shaped alongside PSMα3 self-assembly and promoted on the membrane interface, possible disrupt membrane integrity by way of peptide accumulation and subsequent membrane thinning in a peptide focus and lipid-dependent method. Total, our multiscale and multimodal method sheds new mild on the important thing roles of N-formylation and intermediate self-assembling entities, somewhat than mature fibrils, in dictating deleterious interactions of PSMα3 with membrane lipids, possible underscoring its final mobile toxicity in vivo, and in flip S. aureus pathogenesis.