The production of Nile tilapia (Oreochromis niloticus) has good technological development; however, today, it is still necessary to make it more efficient. One way to increase efficiency is to prevent disease and improve the food conversion factor. Since previous investigations of tilapia microbiota detected a high proportion of organisms belonging to the order Actinomycetes, this study was to isolate, identify, and describe the species of bacteria microbiota belonging to the cultured Nile tilapia. These were done with Nile tilapia grown in a warm sub-humid climate during spring and summer seasons. The biopsy of different organs was performed for bacteriological culture and 16S rRNA sequencing analysis. From the 180 tissue samples, 49 isolates of the order Actinomycetes were obtained, representing ten species from seven genera: Microbacterium, Brevibacterium, Cellulomonas, Corynebacterium, Kocuria, Actinomyces, and Micrococcus. In spring, Microbacterium dominated, accounting for 74% of the total population. In the summer, lower diversity was observed, with 39% represented by Microbacterium. 16S rRNA sequencing analysis enabled the classification of Actinomyces neuii and Microbacterium lacticum as Kocuria varians and Agromyces indicus; the classification of Microbacterium imperiale as Rhodococcus and Micrococcus luteus was confirmed. No sequences of K. varians have been reported in fish. Microbacterium dextranoliticum showed high similarity to environmental samples. Here is the first study that analyzes the bacteria population in tilapia at the genetic level with an ecosystem approach, present in healthy cultured tilapia, indicating their beneficial associations with the host, making them candidates as probiotics, among other possible functions, applicable in tilapia cultivation.