Bacterial diseases ra large problem in aquaculture hatcheries. The successful design and implementation of protective measures in the hatchery depends on an understanding of the dynamics of the infection process. Developing an in situ experimental protocol for pathogen challenge is therefore of paramount importance. Here, we demonstrated the minimum effective pathogenic dose (MEPD) of Vibrio splendidus (105 CFU ml-1) and a Vibrio coralliilyticus/neptunius-like isolate, Vibrio sp. DO1 (106 CFU ml-1), for New Zealand Greenshell™ mussel (GSM, Perna canaliculus) larvae during hatchery production. In a flow-through water hatchery system, larvae given 1 to 2 h of static water exposure to these pathogen doses showed respective average cumulative mortalities of 58 and 69% on the fourth day following pathogen exposure. After the 1 to 2 h static exposure, larvae were returned to flow-through water. Larvae exposed to a dosage one order of magnitude greater than the MEPD had higher mortalities of 73 and 96% for V. splendidus and Vibrio sp. DO1 respectively. These 4 levels of mortality were significantly greater than those of the non-exposed control larvae which respectively averaged 23 and 35% in experiments involving V. splendidus and Vibrio sp. DO1. Experiments were repeated 4 times to confirm reproducibility. After pathogen exposure, pathogens were detected in the larvae and tank water of treatments with dosages of ≥ 105 CFU ml-5 (V. splendidus) and 106 CFU ml-1 (Vibrio sp. DO1), but not in treatments with lower pathogen dosages. The challenge protocols are reproducible and provide an opportunity to assess measures for the protection of GSM larvae against infection in the hatchery environment.