Biological membranes are complex assemblies of lipids and proteins that maintain cellular integrity and homeostasis. Bacterial pore-forming toxins (PFTs) compromise host cell membrane barrier by creating nanoscale pores. While membrane-mimetic models have been widely used to study PFT behaviour, their interactions with live cell membranes remain underexplored. This study investigates the pore-forming mechanism of YaxAB, a bipartite PFT from Yersinia enterocolitica, to elucidate its stepwise assembly and membrane disruption dynamics. YaxAB consists of two subunits: YaxA, which independently binds to the membrane, and YaxB, which facilitates pore formation and cell lysis. Structural studies suggest that YaxA-YaxB dimers assemble into decameric transmembrane pores, but the exact sequence of events leading to pore formation remains unclear. Using live-cell, single-molecule imaging in HeLa cells, we demonstrate that the addition of YaxA followed by YaxB induces cellular blebbing upon pore formation. YaxA alone or YaxA and YaxB together exhibited heterogeneous anomalous diffusion on the membrane, likely influenced by lipid nanodomains and cortical actin organisation. Contrary to previous assumptions that YaxA remains monomeric on the membrane, we observed that YaxA molecules merged to form ’pre-pores’, which rapidly trigger membrane disruption upon YaxB addition. Taken together, we propose a novel mechanism wherein soluble YaxA first binds the membrane, self-assembles into pre-pores, and subsequently recruits YaxB to form functional transmembrane pores leading to ion homeostasis disruption and cell lysis.