Enterotoxigenic Escherichia coli (ETEC), a predominant pathogen responsible for neonatal animal diarrhea, poses a significant threat to global health and results in considerable economic losses in animal husbandry. The pathogenicity of ETEC is primarily mediated by its adherence to intestinal epithelial cells via fimbriae adhesins and the subsequent secretion of enterotoxins. We developed a high-throughput screening platform for chorionic inhibitors. By employing semi-solid motility assays, hemagglutination inhibition assays, and cell adhesion assays, we screened a panel of natural compounds and identified Isogliquiritin (ISL) as the active compound. Subsequent validation using transmission electron microscopy and reverse transcription-polymerase chain reaction (qRT-PCR) revealed that ISL significantly inhibits the synthesis and assembly of ETEC fimbriae, thereby reducing bacterial adhesion to host cells. After assessing its safety, we demonstrated through in vivo experiments that ISL attenuates ETEC-induced diarrhea. In the Galleria mellonella larval model, ISL increased the survival rate, meanwhile in a mouse diarrhea model, ISL reduced inflammation caused by ETEC C83902 and upregulated the expression of tight junction protein (ZO-1, claudin-1, and occluding) genes. Collectively, our findings highlight that ISL is a promising natural compound for the prevention and treatment of ETEC-induced diarrhea. By targeting bacterial virulence factors rather than bacterial growth, ISL provides a novel strategy against antimicrobial resistance and offers a safer alternative for managing ETEC infections in livestock.