Listeriolysin-O (LLO) plays a crucial role during infection by Listeria monocytogenes. It enables escape of bacteria from phagocytic vacuole, which is the basis for its spread to other cells and tissues. It is not clear how LLO acts at phagosomal membranes to allow bacterial escape. The mechanism of action of LLO remains poorly understood probably due to unavailability of suitable experimental tools that could monitor LLO membrane disruptive activity in real time. Here, we used high-speed atomic force microscopy (HS-AFM) featuring high spatio-temporal resolution on model membranes and optical microscopy on giant unilamellar vesicles (GUVs) to investigate LLO activity. We analyze the assembly kinetics of toxin oligomers, the prepore-to-pore transition dynamics and the membrane disruption in real time. We reveal that LLO toxin efficiency and mode of action as a membrane-disrupting agent varies strongly depending on the membrane cholesterol concentration and the environmental pH. We discovered that LLO is able to form arc pores as well as damage lipid membranes as a lineactant, and this leads to large-scale membrane defects. These results altogether provide a mechanistic basis of how large-scale membrane disruption leads to release of Listeria from the phagocytic vacuole in the cellular context. Listeriolysin-O (LLO) plays a crucial role in Listeria monocytogenes infection by allowing bacteria to escape from intracellular phagosomes and cells via an unknown molecular mechanism. We used high-speed atomic force microscopy (HS-AFM) supported with giant unilamellar vesicles imaging (GUVs) to characterize the interaction and dynamics of LLO with the lipid membranes at the nano-and micro-scale. We show that LLO efficiency and mode of action as a membrane-disrupting agent is strongly dependent on membrane cholesterol content and environmental pH. LLO is able to form arc pores and damage membranes as a lineactant, which is crucial for the processive membrane disruption. The latter mechanism, a previously uncharacterized mode of action for this toxin, is strongly cholesterol dependent and may provide a novel angle of attack against listeriosis.