Background: Escherichia coli O157:H7 is a significant pathogen responsible for causing diarrhea in humans. Oxadiazoles are known for their diverse range of biological activities. Objectives: This study aimed to assess the anti-E. coli O157:H7 effects of 1, 3, and 4-oxadiazole derivatives. Methods: Compounds containing an oxadiazole central core were synthesized anew. In vitro assays, including agar well diffusion, minimum inhibitory concentration, and minimum bactericidal concentration, were conducted. The molecular structures of the oxadiazole derivatives were optimized using the mm2 methodology with Chem3D v20.1.1.125 software. The ligand's inhibitory potential against the active sites of stx-1 and stx-2 was assessed using Autodock Vina software. The results were analyzed using Discovery Studio v16.1.0 software. Results: The findings indicated that compound C ((2E)-3-(3,4-difluorophenyl)-2-(5-(hydroxy(pyridin-2-yl)meth yl)-1,3,4-oxadiazol-2-yl)-N-methylacrylimidic acid) exhibited more potent anti-E. coli O157:H7 effects compared to other compounds and the control sample. Furthermore, in silico results demonstrated that compound C exhibited inhibitory effects against stx-1 and stx-2 by forming hydrogen bonds for inhibition. Conclusions: Compounds featuring a fluorophenyl structure with a 1, 3, and 4-oxadiazole core have the potential to serve as anti-E. coli O157:H7 agents for the development of therapeutic drugs.