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We investigated the effects of skatole (3-methylindole), which is one of the indole derivatives on the biofilm formation of EHEC O157:H7. Notably, skatole (100 μg ml−1) significantly reduced EHEC O157:H7 ATCC 43894 biofilm formation by 52% in 96-well polystyrene plates under quiescent conditions, with no effect on planktonic cell growth. The skatole sample was maintained in stable conditions for 24 h without degradation or evaporation via EHEC O157:H7 ATCC 43894. Importantly, skatole negatively triggered the expression of catalase in EHEC strains, as well as altered EHEC surface morphology. Our finding indicated that suppressed catalase activity via skatole might have been responsible for elevated endogenous oxidative stress and increment in oxidative metabolites might have led to damaged cell surfaces and a reduction in biofilm formation of EHEC O157:H7 ATCC 43894.Significance and Impact of the Study: Our findings suggest that inefficient catalase activity of skatole-exposed enterohemorrhagic Escherichia coli (EHEC) O157:H7 ATCC 43894 may account for elevated endogenous oxidative stress, leading to damaged cell surfaces and reduction in biofilm formation. Our results also provide that skatole as a new candidate for bacterial signalling may be applied for inhibiting bacterial biofilms in food and feed industry.