In this study, the identification and characterization of potential probiotic bacteria present in natural microbiota of tambaqui Colossoma macromum were performed for the first time. Bacteria from the intestinal mucosa were isolated in agar Man, Rogosa and Sharpe (MRS). Only Gram‐positive and haemolysis‐negative isolates were selected (n = 152). The tests included morphological differentiation, gene sequencing, catalase, bile tolerance, pathogen antagonism, antimicrobial susceptibility and safety to the host. To differentiate the isolates, colonies were morphologically characterized. From 152 initial isolates, 6 strains with probiotic potential were selected and identified through sequencing of 16S rRNA gene of Enterococcus faecalis, Enterococcus hirae, Lactococcus lactis, Pediococcus pentosaceus, Staphylococcus hominis and Staphylococcus saprophyticus. The growth of the six strains was not affected by different levels of bile inclusion. All probiotic strains inhibited the growth of the Lactococcus garvieae pathogen in the antagonism test. When tested against Aeromonas hydrophila, L. lactis and E. hirae strains were prominent. All strains were resistant to some degree against the 11 antibiotics. With this study, it was possible to screen the following six strains with probiotic potential for inclusion in the diet of C. macropomum: S. hominis, S. saprophyticus, L. lactis, P. pentosaceus, E. hirae and E. faecalis.