In recent years the incidence of vibriosis has greatly increased, raising the concern among consumers about the innocuity of certain food products. Previous studies demonstrated various advantages of molecular methods, including qPCR, for the screening of food-borne pathogens. The new method developed in the present study allows fast and reliable detection of the main human pathogenic Vibrio species (Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus). Specificity of the combination of primers and probes was successfully tested against several bacterial species and strains (44 different strains). Evaluation of the qPCR efficiency reported a value of 94% with the simultaneous amplification of the internal amplification control. The evaluation of the quality of the method was based on six parameters: relative sensitivity, specificity, accuracy, positive and negative predictive values as well as Kappa index of concordance. Each of the values obtained were higher than 96%. Additionally a very low limit of detection was determined for the developed method (less than 10 cfu/25 g). All the parameters of the method analyzed were obtained from the analysis of a wide variety of foodstuffs, water samples and reference material from proficiency tests, and compared against the culture reference method.