Background and Objectives: Previous studies have shown that polymyxin B sulfate and trimthoprim antibiotics are not individually effective on Yersinia enterocolitica and their closely related species. The aim of this study was to evaluate the synergistic effect of above antibiotics on Y. enterocolitica and their closely related species, from the clinical and the natural environment specimen collected in Iran, and compare them with the isolates that that were obtained from the Pasteur institute collection in France. Materials and Methods: In total, 73 species from Iran and 25 from the Pasteur institute in France were tested. The microdilution method was used for the MIC according to the standard protocol. Results: The synergistic effect was seen in all tested samples. However, the human species from the Pasteur institute were more sensitive than the Iranian human and the environmental species were less sensitive than clinical. The Y. enterocolitica isolates were less sensitive than the related species such as Y. intermedia, Y. fredriksenii, and Y. kristensenii. Conclusion: The synergistic effect polymyxin B sulfate and trimthoprim were more evident on other closely related Yersinia species Y. enterocolitica.