We present an in-depth electrophysiological analysis of Tse5, a pore-forming toxin (PFT) delivered by the type VI secretion system (T6SS) of Pseudomonas aeruginosa. The T6SS is a sophisticated bacterial secretion system that injects toxic effector proteins into competing bacteria or host cells, providing a competitive advantage by disabling other microbes and modulating their environment. Our findings highlight the dependency of Tse5 insertion on membrane charge and electrolyte concentration, suggesting an in vivo effect from the periplasmic space. Conductance and selectivity experiments reveal a predominant and reproducible pore architecture of Tse5, characterized by a weak cation selectivity without chemical specificity. pH titration experiments suggest a proteolipidic pore structure influenced by both protein and lipid charges, a hypothesis further supported by experiments involving engineered mutants of Tse5 with altered glycine zippers. These results significantly advance our understanding of Tse5's molecular mechanism of toxicity, paving the way for potential applications in biosensing and macromolecular delivery.