Technical Abstract: The use of by-product feedstuffs and prebiotics in animal rations has increased in recent years, but, in general, the effects of these by-products on the microbial ecosystem remain unknown. The present study was undertaken to determine what effects novel by-product feedstuffs, including prebiotics, have on survival of the important foodborne pathogenic bacteria Escherichia coli O157:H7 and Salmonella enterica Typhimurium in pure and mixed ruminal and fecal culture fermentations from cattle and swine. By-product feedstuffs utilized in this study included: hyperimmunized whole egg, lysine biomass, lysine biomass (spray-dried), threonine biomass (drum-dried), threonine biomass (spray-dried), beer well yeast (drum-dried), beer well yeast (spray-dried), ethanol yeast (pan-dried), and corn meal as a control to simulate normal dietary conditions. Prebiotics examined included: PremiDex™, CitriStim™, a CitriStim:PremiDex blend (50%:50%), and a commercial oligosaccharide source feedstuff. Pure culture populations of Escherichia coli O157:H7 were reduced (P < 0.05) by 2% w/v of each of: spray-dried threonine, drum-dried threonine, ethanol yeast, hyper-immunized whole egg, and a blend of CitriStim:PremiDex. Fermentations of mixed ruminal microorganisms from cattle demonstrated that 2% PremiDex reduced (P < 0.05) E. coli O157:H7 populations compared to controls in cattle fed a forage-based ration, and the CitriStim:PremiDex blend reduced E. coli O157:H7 populations (P < 0.05) in fermentations from cattle fed high-grain rations. The anti-foodborne pathogen effect appears to be an indirect effect mediated by the microbial population of the intestinal tract, similar to a probiotic effect. Further research is needed to identify modes of action of these by-product feedstuffs and impacts they have on the mixed intestinal microbial ecosystem.