Ready-to-eat meat products such as dry-fermented sausages have been associated with foodborne outbreaks despite the multiple hurdles used in the manufacturing process to prevent growth of pathogens. As a result, new strategies such as natural products with antimicrobial activity are being used to control pathogens of importance like Escherichia coli O157:H7. This study investigated how different concentrations and sources of mustard can influence its antimicrobial activity against E. coli O157:H7 in dry-fermented sausage, as well as the contribution of residual myrosinase enzyme in mustard to this process. The genetic basis for the degradation of mustard glucosinolate by E. coli O157:H7, which is associated with the antimicrobial action of mustard, was also characterized. The ability of E. coli O157:H7 to withstand inhibitory allyl isothiocyanate (AITC) concentrations and the role of the two-component BaeSR system as a defense mechanism against AITC was also investigated. Results showed that 4% (w/w) deodorized yellow mustard powder was effective to control E. coli O157:H7 in dry-fermented sausage at 28 d. The presence of endogenous plant myrosinase in the mustard powder or meal enhanced E. coli O157:H7 reduction rates. Fully-deodorized, deoiled, yellow mustard meal as low as 2% (w/w) containing either 0.1% or 0.2% of residual plant myrosinase achieved the same results as 4% (w/w) mustard powder also containing similar residual myrosinase. Regardless of the type of mustard, the antimicrobial activity of yellow mustard derivatives were more pronounced than those of Oriental mustard. The initial genetic assessment through in silico analysis found similarity between plant myrosinase and enzymes encoded by genes (bglA, ascB, and chbF) from β-glucosidase families in E. coli O157:H7 strains. After disruption of these genes using lambda-red replacement, single (∆bglA, ∆ascB, ∆chbF) and double (∆bglAascB, ∆chbFascB, ∆chbFbglA) mutant strains were created and assessed for glucosinolate degradation. The comparison of the gene expression profiles and changes in the extent of sinigrin degradation by different mutants suggested that ascB have a prominent role in the degradation of this β-glucoside by E. coli O157:H7. E. coli O157:H7 did not develop resistance to AITC, the essential oil formed from sinigrin degradation that is responsible for the antimicrobial activity of Oriental mustard.