We performed a high-throughput screening (HTS) against Escherichia coli O157:H7 Ler. The HTS system is a rapid and cost-effective tool for probing Ler inhibition. Yomogin specifically inhibits the expression of virulence factors. Yomogin has potential as a novel anti-pathogenic agent for E. coli O157:H7. Yomogin can overcome the issue of antibiotic resistance.