This study aims to mine a previously developed continuous-flow competitive exclusion culture (CFCEC) originating from the Tilapia gut microbiome as a rational and efficient autochthonous probiotic strain recovery source. Three isolated strains were tested on their adaptability to host gastrointestinal conditions, their antibacterial activities against aquaculture bacterial pathogens, and their antibiotic susceptibility patterns. Their genomes were fully sequenced, assembled, annotated, and relevant functions inferred, such as those related to pinpointed probiotic activities and phyloge-nomic comparative analyses to the closer reported strains/species relatives. The strains are possible candidates of novel genus/species taxa inside Lactococcus spp. and Priestia spp. (previously known as Bacillus spp.) These results were consistent with reports on strains inside these phyla exhibiting probiotic features, and the strains we found are expanding their known diversity. Furthermore, their pangenomes showed that these bacteria have indeed a set of so far uncharacterized genes that may play a role in the antagonism to competing strains or specific symbiotic adaptations to the fish host. In conclusion, CFCEC proved to effectively allow the enrichment and further pure culture isolation of strains with probiotic potential.