We evaluated the performance and cost-effectiveness of a matrix-assisted laser desorption ionization time-of-flight mass spectrometry-based Biotyper system for the routine identification of common enteric bacterial pathogens seen in middle Tennessee from suspicious colonies grown on selective stool culture media. A total of 304 suspicious colonies were selected and further identified from 605 stool specimens. The suspicious colonies were analyzed by the Biotyper system, and the results were compared to those from routine phenotypic methods, which identified 22 Salmonella species, 39 Shigella species, 3 enterohemorrhagic Escherichia coli (EHEC) isolates, 2 Yersinia enterocolitica isolates, 2 Campylobacter species, and 236 gastrointestinal normal flora isolates. The Biotyper system correctly identified the Salmonella species, Yersinia enterocolitica, and Campylobacter species but failed to distinguish the Shigella species and EHEC isolates from E. coli. Among the 236 normal flora isolates, 233 (98.7%) and 228 (96.6%) agreed at the genus and species levels, respectively, between the phenotypic and Biotyper methods. Organism identification scores were insignificantly different between colonies directly from selective media and subsequently from pure subculture. The entire Biotyper identification procedure, from smear preparation to final result reporting, can be completed within 30 min. The Biotyper system provides a rapid and simple screening tool for identifying many, but not all, suspicious colonies grown on selective media within 24 h after inoculation, which shortens test turnaround time by 2 to 3 days.