Enterohemorrhagic Escherichia coli O157: H7 (EHEC), a foodborne enteropathogen, remains a significant public health concern since its discovery in 1982. With an incredibly low infectious dose (10-100 bacteria), this pathogen can cause self-limiting diarrhea, vomiting, and abdominal cramps. However, more complicated disease conditions such as bloody diarrhea or hemolytic colitis have been known to develop depending on the serotype involved in the infection, and on immune status and/or age of the patients. Due to its Shiga toxin (Stx) production ability, EHEC infection may lead to a kidney-related problem known as hemolytic uremic syndrome (HUS), which requires advanced medical care. Unlike other bacterial illnesses, therapeutic administration of antibiotics to treat EHEC infections is not recommended due to their controversial association with Stx production. As a result, only preventative/prophylactic and immune-supportive strategies are followed for EHEC infections. Using the antibacterial properties of probiotic bacteria and the metabolites they produce are promising alternative strategies for preventing EHEC infections. We have targeted the probiotic bacteria Lactobacillus casei to determine the mechanism of this alternative strategy. In our study, we have executed microbiological, molecular, chromatographic, and metagenomic approaches to determine the antagonistic mechanisms of action of their metabolites, specifically conjugated linoleic acid (CLA) produced by Lactobacillus casei, against the growth and metabolism of EHEC. The metabolites of wild-type L. casei (LCwt) were augmented by supplementing it with a prebiotic-like dietary component, namely peanut flour (PF) (LCwt+PF), while another LCwt was also genetically engineered (LCCLA) to over convert CLA from linoleic acid (LA). These modifications showed effective results in controlling EHEC both in vitro and in ex vivo conditions. Total metabolites present in cell-free culture supernatant (CFCS) of LCwt, LCwt+PF, and LCCLA were able to control the growth of EHEC without negatively hampering the relative abundance of Firmicutes and Bacteroidetes present in rumen fluid (RF). Among these CFCSs, CFCSCLA exerted the most desirable outcome by eliminating EHEC. In vitro studies demonstrated that, a lower concentration of purified CLA worked synergistically with other metabolites of LCwt and augmented their inhibitory activity against EHEC. The orchestrated effect of metabolites has been observed to downregulate the virulence genes, disrupt the cell membrane, interfere with cell division, and damage their genomic DNA. The probable effect of these metabolites, specifically CLA, on Stx production and neutralization was also investigated by assessing host cell cytotoxicity. Total metabolites of Lactobacillus spp. as well as CLA itself, showed improvement in cell survivability when exposed to Stx. Our findings established a ground to explore the effect of specific metabolites obtained from probiotic bacteria in control and prevention of EHEC. The findings also showed a promising association of purified CLA in neutralizing Stx which can be further explored to use it in therapeutic purposes.