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Pore-forming toxins (PFTs) are recognized as major virulence factors produced by both Gram-positive and Gram-negative bacteria. While the effects of PFTs have been extensively investigated using mammalian cells as a model system, their interactions with the environmental host, Acanthamoeba castellanii remains less understood. This study employed high-throughput image screening (HTI), advanced microscopy, western blot analysis, and cytotoxicity assays to evaluate the impact of PFT-producing bacterial species on their virulence against A. castellanii. Our unbiased HTI data analysis reveals that the cyst induction of A. castellanii in response to various bacterial species does not correlate with the presence of PFT-producing bacteria. Moreover, A. castellanii demonstrates resistance to PFT-mediated cytotoxicity, in contrast to mammalian macrophages. Notably, Vibrio anguillarum and Ralstonia eutropha triggered a high frequency of cyst formation and cytotoxicity in infected A. castellanii. In summary, our findings reveal that A. castellanii exhibits a unique resistance to PFTs, unlike mammalian cells, suggesting its potential ecological role as a reservoir for diverse pathogenic species and its influence on their persistence and proliferation in the environment. (Figure presented.)