The objective of this study was to isolate from chickens potential competitive exclusion bacteria (CE) that are inhibitory to Campylobacter jejuni or Salmonella, or to both, for subsequent development of a defined CE product for use in poultry. Adult chickens from family farms, commercial farms, and broiler chicken research centers were sampled to identify and select C. jejuni-free donor chickens. A challenge treatment, which included administering perorally 106 CFU C. jejuni per chicken and determining undetectable cecal shedding of campylobacters at 4 weeks, was important for identifying the best CE donor chickens. Screening of bacterial colonies obtained from nine donor chickens by using selective and nonselective media yielded 636 isolates inhibitory to six C. jejuni strains in vitro, with 194 isolates being strongly inhibitory. Of the 194 isolates, 145 were from ceca, and 117 were facultative anaerobic bacteria. One hundred forty-three isolates were inhibitory to six strains of Salmonella (including five different serotypes) in vitro. Of these, 41 were strongly inhibitory to all C. jejuni and Salmonella strains evaluated, and most were Lactobacillus salivarius. A direct overlay method, which involved directly applying soft agar on plates with discrete colonies from mucus scrapings of gastrointestinal tracts, was more effective in isolating CE than was the frequently practiced isolation method of picking and transferring discrete colonies and then overlaying them with soft agar. The best approach for obtaining bacteria highly inhibitory to Salmonella and C. jejuni from chickens was to isolate bacteria from ceca under anaerobic conditions. Free-range chickens from family farms were better donors of potential CE strongly inhibitory to both Salmonella and Campylobacter than were chickens from commercial farms and broiler chicken research centers.