AimsThe aim of this study was to determine the intestinal microbiota of pirarucu (Arapaima gigas) in different growth stages (adult and fingerlings) and to isolate and identify potential probiotic bacteria. Methods and ResultsHigh-throughput sequencing analysis of the intestinal contents revealed that the majority of sequences belonged to the Proteobacteria, Fusobacteria and Firmicutes phyla. At the genus level, the greatest number of sequences belonged to Bradyrhizobium in adult fish, while Cetobacterium was the most abundant in juvenile fish. Twenty-three lactic-acid bacteria (LABs) were isolated on MRS agar from healthy juvenile fish. The isolates were tested invitro for probiotic properties. Two isolates (identified as strains of Lactococcus lactis subsp. lactis and Enterococcus faecium) displayed antagonism against all 10 pathogens tested, were nonhaemolytic and maintained good viability for at least 3weeks when supplemented to fish diets. The presence of a number of antibiotic resistance genes (ARGs), conferring resistance to erythromycin, tetracycline and chloramphenicol, was investigated by PCR. ConclusionsThe absence of ARGs investigated the potential to antagonize pathogens, and favourable growth and survival characteristics indicate that these autochthonous isolates have the potential to be considered probiotics, which will be studied in future invivo experiments. Significance and Impact of the StudyThis study has demonstrated, for the first time, the normal microbiota in the A. gigas intestine during different life stages and the presence of LAB strains. It also demonstrated LAB antibiotic resistance and antagonistic behaviour against pathogens isolated from the same fish.