This study investigated the antimicrobial and antibiofilm effects of 16 halogenated indoles (fluoro-, chloro-, bromo-, or iodo-) and indole against Escherichia coli O157:H7 (EHEC). The antibiofilm activity followed the order chloroindoles > bromoindoles > indole > fluoroindoles. The minimum inhibitory concentrations (MICs) of 4-bromoindole and 5-bromoindole were 100 and 200 μg/mL, respectively. At 20 μg/mL, both compounds inhibited EHEC biofilm formation by more than 61% without affecting planktonic cell growth. However, at concentrations above their MICs, both showed bactericidal activity. Scanning electron microscopy confirmed the antibiofilm effects, showing that both 4-bromoindole and 5-bromoindole reduced swimming and swarming motility and curli formation, which are important for EHEC biofilm formation. Quantitative structure–activity relationship analysis showed that halogenation at positions C-4 or C-5 promotes antimicrobial activity, while substitution at C-7 is detrimental. The study suggests that halogenated indoles, particularly bromoindoles, have potential as antimicrobial and antibiofilm agents against EHEC. EHEC is a pathogenic bacteria that forms biofilms on various surfaces, making it a challenge for treatment. The formation of biofilms reduces drug sensitivity and contributes to bacterial persistence in chronic infections. Indole and its derivatives act as bacterial signaling molecules and can suppress EHEC virulence and biofilm formation. Many halogenated indoles have demonstrated antimicrobial and antibiofilm effects on other microorganisms. This study systematically investigated the antibacterial and antibiofilm activities of 16 halogenated indoles against EHEC, using quantitative structure–activity relationship (QSAR) assays to determine which substitutions most effectively enhanced antimicrobial actions. The mechanisms of action were explored through growth kinetics, scanning electron microscopy (SEM), as well as motility and curli assays. The results indicate that bromoindoles are effective in inhibiting EHEC biofilm formation and growth.This study investigated the antimicrobial and antibiofilm effects of 16 halogenated indoles (fluoro-, chloro-, bromo-, or iodo-) and indole against Escherichia coli O157:H7 (EHEC). The antibiofilm activity followed the order chloroindoles > bromoindoles > indole > fluoroindoles. The minimum inhibitory concentrations (MICs) of 4-bromoindole and 5-bromoindole were 100 and 200 μg/mL, respectively. At 20 μg/mL, both compounds inhibited EHEC biofilm formation by more than 61% without affecting planktonic cell growth. However, at concentrations above their MICs, both showed bactericidal activity. Scanning electron microscopy confirmed the antibiofilm effects, showing that both 4-bromoindole and 5-bromoindole reduced swimming and swarming motility and curli formation, which are important for EHEC biofilm formation. Quantitative structure–activity relationship analysis showed that halogenation at positions C-4 or C-5 promotes antimicrobial activity, while substitution at C-7 is detrimental. The study suggests that halogenated indoles, particularly bromoindoles, have potential as antimicrobial and antibiofilm agents against EHEC. EHEC is a pathogenic bacteria that forms biofilms on various surfaces, making it a challenge for treatment. The formation of biofilms reduces drug sensitivity and contributes to bacterial persistence in chronic infections. Indole and its derivatives act as bacterial signaling molecules and can suppress EHEC virulence and biofilm formation. Many halogenated indoles have demonstrated antimicrobial and antibiofilm effects on other microorganisms. This study systematically investigated the antibacterial and antibiofilm activities of 16 halogenated indoles against EHEC, using quantitative structure–activity relationship (QSAR) assays to determine which substitutions most effectively enhanced antimicrobial actions. The mechanisms of action were explored through growth kinetics, scanning electron microscopy (SEM), as well as motility and curli assays. The results indicate that bromoindoles are effective in inhibiting EHEC biofilm formation and growth.