Bordetella pertussis/ parapertussis/ bronchiseptica DNA was identified from nasopharyngeal samples and/or culture using a multiplex Real-Time Polymerase Chain Reaction (PCR) method. DNA was extracted from clinical samples and culture, in case of bacterial growth and results were compared. A conventional PCR was also performed on DNA extracted from culture and ptxP gene encoding for pertussis toxin promoter was confirmed for B. pertussis strains. 14 out of the 26 nasopharyngeal samples tested by multiplex Real-Time PCR were positive for B. pertussis and for the 6 isolated strains. B. parapertusis and B. bronchiseptica were not identified in the clinical samples; however Cycle threshold (Ct) values were obtained in the case of DNA extracted from reference strains included in the study for optimization of the protocol. Multiplex PCR reaction proposed in this study is an adequate method that can be used in the molecular diagnostic of whooping cough, which can be performed directly on DNA extracted from pathological samples.