Aims:The aim of this study was to analyze the physiological properties, pathogenicity and application of probiotic bacteria to catfish (Clarias sp.) cultivation in vivo. Methodology and results: Probiotic bacteria's physiological characteristics were described using descriptive approaches, whereas experimental methods analyzed pathogenicity tests and the application of the probiotic bacteria in catfish cultivation. The results of bacterial characterization showed that Bacillus safensis strain C251 SA3, Bacillus amyloliquefaciens strain NO10 SA8 and Bacillus australimaris strain IIHR GAPB01 SL1 had bacili cell shape and showed a positive catalase test for all test bacteria except Bacillus safensis strain C251 SA3. The three probiotic bacteria also had negative oxidative-fermentative results, which were able to reduce nitrate and survive in environmental conditions with pH 3-9. Pathogenicity analysis revealed that the three bacteria had the potential to be non-pathogenic or less pathogenic. Bacillus safensis strain C251 SA3 treatment on catfish resulted in the best survival rate of 64% and a 17.5 cm growth in catfish body length by week 13. When Bacillus amyloliquefaciens strain NO10 SA8 was applied to catfish, the survival rate was 32% and the catfish body length increased by 22 cm by week 13. The control treatment revealed that all catfish samples died or had a 0% survival rate, while the application of Bacillus australimaris strain IIHR GAPB01 SL1 revealed a catfish survival rate of 0%. Conclusion, significance and impact of study: Bacillus australimaris strain IIHR GAPB01 SL1 is a potential probiotic bacterium to be applied in catfish food.