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Antimicrobials have been important medicines used to treat various infections. However, some antibiotics increase the expression of Shiga toxin (Stx). Also, the pervasive use of persistent antibiotics has led to ecotoxicity and antibiotic resistance. In this study, a newly developed broad-spectrum and reversible antibiotic (guanylhydrazone disinfectant) was evaluated for its antibiotic activity and effects on Stx production and global transcription of bacteria. No Stx induction was observed in 25 Shiga toxin-producing E. coli (STEC) isolates treated with a sublethal concentration of the guanylhydrazone. A differential gene expression study comparing two guanylhydrazone-treated to non-treated E. coli strains indicated that the expression of a group of stress-responsive genes were enhanced. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that guanylhydrazone treatment significantly downregulated the pathways of ribosome and flagellar assembly in both pathogenic and non-pathogenic strains and differentially regulated some pathways essential for bacteria to maintain cell shape and gain survival advantage in two strains. In addition, upregulation of antibiotic resistant genes related to the multidrug efflux system and virulence genes coding for colibactin, colicin, and adhesin was observed in strains treated with the disinfectant. The knowledge obtained in this study contributes to our understanding of the mode of this disinfectant action and facilitates our effort to better use disinfectants for STEC treatments. Full article