Concerns over the development of antibiotic-resistant bacteria within the food animal industry have intensified the search for natural approaches to the prevention and treatment of bacterial diseases. Competitive exclusion cultures are the foundation of a disease-management strategy based on the use of benign bacterial strains to prevent the establishment of pathogenic bacteria within a specific host. Differentiation of phenotypically ambiguous isolates is a critical step in establishing a manageable library of bacteria for use in the development of defined competitive exclusion cultures. We used automated ribotyping techniques to dereplicate a large collection of phenotypically ambiguous isolates from a continuous-flow competitive exclusion culture. A total of 157 isolates were screened following an EcoRI restriction enzyme digestion. The 157 isolates were resolved into 23 ribogroups, which represents an 85% reduction in the number of isolates in the bacterial isolate library. Seventy-six percent of the isolates fit into one of five ribogroups. This work demonstrated that automated ribotyping is an effective and efficient tool for dereplication of diverse bacterial isolate libraries.