In New Caledonia, shrimp hatcheries are confronted with mass mortality in the larval stages, a phenomenon poorly understood as no specific causative agent has been identified. This has resulted in an excessive use of prophylactic antibiotics, although their adverse effects in aquaculture are notorious. The present work was thus aimed at selecting potential probiotic strains for penaeid hatcheries. From a pool of more than 400 marine bacterial isolates sampled from the local marine environment seven strains exhibited in vitro antagonistic activity towards Vibrio harveyi. These isolates were characterized both phenotypically and genotypically using a biochemical approach and 16S rDNA sequencing. Six out of these seven strains were found to belong to the genus Pseudoalteromonas, the last one belonging to the Vibrionaceae family and related to the Harveyi clade. Selected probiotic candidates were individually tested for antagonistic activities in vitro using a green fluorescent protein (GFP)-labelled transconjugant of V. harveyi and for inherent pathogenicity towards cultured shrimp larvae at two different developmental stages. This approach enabled rapid processing and selection of candidates to be tested in our experimental hatchery. Four different experiments were conducted to test candidate strains either alone or in combination. Repeated trials showed that postlarval survival was significantly improved by adding the strain NC201 individually to the rearing water compared to unchallenged controls. The analysis of immune-related gene expressions showed that the Litsty PEN3 transcript abundance of larvae was significantly increased after being reared in probiotic-containing water; however no significant difference in lysozyme gene expression was recorded in this study. Together, these results open new insights into the use of these strains as potential substitutes to antibiotherapy in shrimp larval rearing in New Caledonia.