A real-time polymerase chain reaction (PCR) assay was developed for the identification and quantification of two oyster species: Ostrea edulis and Crassostrea gigas. Two sets of primers and TaqMan-MGB probes were designed, based on partial sequences of the 16S rRNA gene. An amplification positive control system was also located in the 18S rRNA gene sequences. Closely related species of oysters and other bivalves, known to co-occur with the target species in European waters, were used to test the assay for cross-reactivity. The assay designed was specific for the target species and no signal or no significant signal was detected for all non-target species tested. The high sensitivity of this method was demonstrated since it is possible to detect just one larva (150–200 μm size) of each species even when it is present with others. Furthermore, this assay provided an acceptable quantification of the number of spiked larvae (1, 10 and 100 larvae) in plankton samples employing a standard curve for larvae.