As the number of multidrug resistant pathogens rise, the search for new antibiotics is crucial. Antimicrobial peptides (AMPs) have received attention to fill this pharmacological void as these small host defense molecules induce selective membrane lytic activity against microbial pathogens. The ability of AMPs to target the microbial membrane over that of a host’s has naively been based on the electrostatic attraction of these predominately cationic peptides for the negatively charged microbial membrane. Despite several decades of intense research their application as therapeutic treatments have been hampered from a clear understanding of their universal mechanism of interaction, given these molecular species vary widely in terms of both their primary sequence and secondary structures making structure-function relationships difficult. We have previously shown with atomic force microscopy (AFM) that zwitterionic membranes display concentration-dependent structural transformations induced by Protegrin-1 (PG-1)—an 18-residue, cationic, β-sheet AMP isolated from pig leukocytes—that display finger-like instabilities at bilayer edges, to the formation of transmembrane pores, and finally to a network of worm-like micelles. The peptide-induced disruption beyond pores of a static size has suggested that AMPs act to lower the interfacial energy of the bilayers in a way similar to detergents. The increasing degree of membrane disruption in charge-neutral membranes demonstrates that a more complex interaction than that suggested by a simple electrostatic argument is needed to explain AMP selectivity in general. I have proposed that in addition to an electrostatic element, specific membrane compositional differences between host and pathogen tunes AMP activity to selectively disrupt microbial membranes. The presence of cholesterol in eukaryotic cell membranes is one of the crucial differences between host and bacterial cell membranes, which evince none. Given that cholesterol stiffens fluid membranes, AMP selectivity may in part be the result of differing membrane fluidities. Isothermal titration calorimetry (ITC) measurements were conducted to characterize PG-1’s affinity to vesicles with increasing cholesterol percentages and has shown decreasing insertion which is ultimately negated by 30 mole% cholesterol. Corollary AFM imaging has supported that PG-1 is less able to insert into cholesterol-containing membranes, as a reduction in pore density and overall disruption was observed. Contrastingly, X-ray grazing-incidence diffraction measurements show that increasing monolayer fluidity, through disruption of the packing of rigid lipid monolayers, can instead enhance PG-1’s insertion, confirming PG-1’s behavior is driven by the mechanical response of the membrane. Cholesterol’s unique ability to impart greater membrane cohesion results in thicker membranes that potentially affects the hydrophobic matching between membrane and peptide, reducing favorable peptide insertion that makes the membrane more resistive. Through oriented circular dichroism studies we effectively showed that when PG-1 was pre-incorporated into mixed membranes containing cholesterol, PG-1’s membrane orientation was quite plastic and indeed confirmed hydrophobic matching can be a regulating factor in PG-1’s activity. Combined AFM and ITC measurements have shown that given membranes of the same relative fluidity, membranes composed of lipids with longer acyl chain lengths are more resistant to the action of PG-1. Through the study of thirteen AMPs that differ in charge, sequence, and secondary structure from across a variety of living species, we show that a common physical ability for the peptides to lower line tension unites their interaction with membranes. While the line-active behavior was not driven by the overall charge of the peptide, the observed line activity was largely correlated with the adoption of imperfect secondary structures, generated by poor amphiphilic residue segregation or breaks in ideal secondary motifs, that commonly positioned charged residues near the membrane interface to favorably induce membrane deformation. The observed universal detergent-like behavior for AMPs makes a fluidity argument for selectivity much more general for AMPs of all charges and potentially implicates other physical attributes of membrane, e.g., lipid spontaneous curvature, in regulating AMP activity.