Probiotics used in aquaculture are mostly from non-fish sources, as a result ineffective in eliciting the desired effects in aquatic animals. In this study, three Bacillus species were isolated from the digestive tract of freshwater fish Oreochromis niloticus and characterised based on their morphological, biochemical and evolutionary relationships. Their probiotic potentials were evaluated based on their ability to tolerate low pH, bile salt concentration, high temperatures, adhesion ability (auto-aggregation and hydrophobicity), haemolytic activity and antimicrobial activity including biosafety assay. Three Bacillus strains identified as Bacillus velezensis TPS3N (MK130897), Bacillus subtilis TPS4 (MK130899) and Bacillus amyloliquefaciens TPS17 (MK130898) were designated as TPS3N, TPS4 and TPS17, respectively. TPS3N and TPS17 were α-haemolytic, while TPS4 was γ-haemolytic. The three isolates had higher viability ability after exposure to higher temperatures (80 °C, 90 °C and 100 °C) and were resistant to low pH (1) and bile salt concentration (0.5%) as well as high cell surface hydrophobicity and autoaggregation. The three isolates were compatible with one another and thus can be used in consortia. These strains were susceptible to gentamicin, cephalexin, ampicillin, ceftriaxone, kanamycin, amikacin, penicillin, cefoperazone, chloramphenicol, erythromycin, tetracycline, doxycycline, ciprofloxacin, clindamycin (except TPS4) and furazolidone (except TPS17). The antimicrobial assessment showed that among the three isolates, TPS3N and TPS17 exhibited good antimicrobial activity against the three fish pathogens (Streptococcus agalactiae, Aeromonas hydrophila, Vibrio harveyi), while TPS4 was effective against Streptococcus agalactiae only. The results of this work suggest that Bacillus strains TPS3N, TPS4 and TPS17 could be considered as potential probiotics in tilapia aquaculture.