Gastrointestinal microbiota have an important impact on fish health and disease, stimulating interest in a better understanding of how these gastrointestinal microbial communities are composed and consequently affect host fitness. In this respect, probiotic microorganisms have been extensively used in recent aquaculture production. To study the use of probiotics in the treatment of infectious diseases, the establishment of a method of experimental infection to obtain consistent results for mortality and infection in challenge tests is important. In pathogen-screening tests, 4 candidate pathogenic bacteria strains (Edwardsiella ictaluri gly09, E. ictaluri gly10, E. tarda LMG2793 and Streptococcus agalactiae LMG15977) were individually tested on xenic Nile tilapia larvae. Only Edwardsiella strains delivered via Artemia nauplii, with or without additional pathogen delivery via the culture water, led to increased mortality in fish larvae. A gnotobiotic Nile tilapia larvae model system was developed to provide a research tool to investigate the effects and modes-of-action of probiotics under controlled conditions. A double disinfection procedure using hydrogen peroxide and sodium hypochlorite solution was applied to the fish eggs, which were subsequently incubated in a cocktail of antibiotic and antifungal agents. In the gnotobiotic challenge test, E. ictaluri gly09R was added to the model system via Artemia nauplii and culture water, resulting in a significant mortality of the gnotobiotic fish larvae. The developed gnotobiotic Nile tilapia model can be used as a tool to extend understanding of the mechanisms involved in host-microbe interactions and to evaluate new methods of disease control.