Ruminants are important reservoirs of E. coli O157:H7 and are considered as the major source of most foodborne outbreaks (e.g., 2017 outbreak in Germany, 2014 and 2016 outbreaks in United States, all linked to beef products). A promising strategy to reduce E. coli O157 is using antimicrobials to reduce the pathogen levels and/or virulence within the animal gastrointestinal tract and thus foodborne disease. The aim of the study was to determine the efficacy of a commercial mixture of natural antimicrobials against E. coli O157. The minimum inhibitory concentration and minimum bactericidal concentration of the antimicrobial were quantitatively determined and found to be 0.5% and 0.75% (v/v) of the natural antimicrobial, respectively. Microbial growth kinetics was also used to determine the effect of the antimicrobial on the pathogen. The natural antimicrobial affected the cell membrane of E. coli O157, as demonstrated by the increase in relative electric conductivity and increase in protein and nucleic acid release. The antimicrobial was also able to significantly reduce the concentration on E. coli O157 in a model rumen system. Biofilm assays showed that subinhibitory concentrations of the antimicrobial significantly reduced the E. coli 0157 biofilm forming capacity without influencing pathogen growth. In addition, the natural antimicrobial was able to reduce motility and exopolysaccharide production. Subinhibitory concentrations of the antimicrobial had no effect on AI-2 production. These findings suggest that the natural antimicrobial exerts an antimicrobial effect against E. coli O157 in vitro and in a model rumen system and could be potentially used to control this pathogen in the animal gut. The results also indicate that subinhibitory concentrations of the antimicrobial effectively reduce biofilm formation, motility, and exopolysaccharide production.