Background and Objective: Bordetella pertussis is a gram-negative cocobacilli bacterium and etiologic agent of whooping cough that in recent years, the number of its cases is on the rise. The ability of biofilm production helps this bacterium in interference with host immune system, severity of illness and antibiotic sensitivity. Thus, due to the importance of this factor, in this investigation, we tested the ability of Bordetella pertussis isolates in biofilm FORMATION.Materials and Methods: Nasopharyngeal samples were collected from persons who suspected whooping cough from throughout of Iran from 2014 to 2016 and were transferred to pertussis reference laboratory in Pasteur Institute of Iran. The bacterium in question was isolated by culture so that finally we had 20 positive samples. At the end, we performed in vitro biofilm FORMATION test for isolated strains.Results: All of the 20 clinical samples of Bordetella pertussis which in this study were examined could form biofilm and their final OD became between 0.606 and 1.212, the power of the biofilm made by them was intermediate. Conclusion: Findings of this study was similar to researches conducted by other researchers, which showed the ability of the strains of Bordetella pertussis to form biofilm and to create adverse effects on the host for example antibiotic resistance, illness severity, and so on. Thus, considering the importance of this issue, more and broader researches are needed in this area.