Validation of a novel quantitative real-time PCR using TaqMan® minor groove binder (MGB) chemistry is described for sensitive and rapid detection of Vibrio aestuarianus, an increasingly important pathogen of Pacific cupped oyster Crassostrea gigas aquaculture. Primers and TaqMan® MGB hydrolysis probe were designed to specifically amplify a 58bp DNA fragment of the V. aestuarianus dnaJ gene. Real-time PCR selectivity was empirically tested using DNA extracted from isolates of V. aestuarianus and a selection of different aquatic bacterial species, including other Vibrio spp. Theoretical selectivity was assessed through sequence comparison using the NCBI BLAST similarity tool. Quantitative PCR plasmid standards were generated to test assay linearity, amplification efficiency and the limit of quantitation (LOQ), according to International Organisation for Standardisation ISO 16140 validation recommendations. LOQ ranged between 5 and 10 PCR copies, although the detection range extended beyond this with reduced precision. Applied performance was tested using C. gigas samples taken from a selection of Irish aquaculture sites. Increasing levels of V. aestuarianus, accompanied by the development of tissue pathology in examined oysters, were found at 1 site that was sampled repeatedly in 2013. Rapid, sensitive and reproducible detections of V. aestuarianus from C. gigas tissue samples were attained during this validation study with a small sample size, and a practical application for disease management is described.