The aim of the present work was to isolate probiotic bacteria from the intestinal tract of healthy freshwater prawn Macrobrachium rosenbergii and to examine the effect of the isolated probiotic Bacillus vireti 01 in controlling Pseudomonas aeruginosa infection. This is probably the first report on the isolation of probiotic B. vireti 01 from the intestine of M. rosenbergii. The compounds present in B. vireti 01 were identified using GC-MS analysis. The effect of B. vireti 01-incorporated diet on survival and antioxidant enzymes was studied in M. rosenbergii for 2 weeks. Decreased mortality was observed in M. rosenbergii which were administered with the probiotic diet compared to control diet. The antioxidant defence enzymes activities such as SOD, catalase and GSH were analysed in various organs of M. rosenbergii probiotic-treated and control groups. Antioxidant enzyme activities were considerably lowered (p < 0.01) in the muscles, hepatopancreas and gills of prawns infected by P. aeruginosa when compared to that of prawns fed with the probiotic-supplemented diet. The histopathological results suggest that the hepatopancreas, gills and muscles infected with P. aeruginosa were altered structurally. The result of the present work demonstrates that the probiotic B. vireti 01 could be used as a substitute to antibiotics for treating P. aeruginosa infection in prawns. © 2017, Springer Science+Business Media, LLC.