This study reports the characterization of the microbial community composition, and the establishment and dynamics of a continuous-flow competitive exclusion culture (CFCEC) derived from gut microbiomes of Nile tilapia (Oreochromis niloticus) specimens reared on aquaculture farms in Colombia. 16S rRNA gene amplicon Illumina sequencing was used to identify taxonomical changes in the CFCEC microbial community over time. The CFCEC was developed from adult tilapia from two farms in Colombia, and CFCEC samples were collected over two months. The pH varied from 6.25 to 6.35 throughout culturing, while anaerobic and aerobic cell counts stabilized at day 9, at 109 CFU mL-1 and were maintained to day 68. A variation in the CFCEC bacterial composition was observed over time. Cetobacterium was the most abundant in the first two days and coincided with a higher CFCEC supernatant antimicrobial effect against the fish pathogen Streptococcus agalactiae. Antimicrobial activity against S. agalactiae disappeared by day 3. Changes in bacterial composition continued to day 33 with Lactococcus spp. becoming the most abundant member of the community. In conclusion, the study of the CFCEC from intestinal tract of Nile tilapia (Oreochromis niloticus) by 16S rRNA gene sequencing allowed identification of predominant bacterial genera in the continuous-flow competitive exclusion culture exhibiting antibacterial activity against the fish pathogen Streptococcus agalactiae.