The present study aimed to optimize the conditions for antioxidant potential hydrolysate (RPMPH) production from Piaractus brachypomus fish (RBPF) meat using the probiotic Bacillus (SNGC) isolated from the chicken gizzard. The RBPF meat exhibited 79.25% moisture, 14.06% protein, 5.08% fat, and 0.60% ash content. RSM generated optimum fermentative hydrolysis conditions was obtained at 48h fermentative time, 2% sucrose concentration, and 30% RBPF meat concentration for the DH (49.41%), DPPH activity (83.14%), ABTS activity (92.02%), and ferric reducing antioxidant power (273.87 µM TE/ g), and Fe²⁺ chelating) activity (68.87%). Furthermore, RPMPH displayed strong antibacterial properties against L. monocytogenes and S. aureus, while they were weak against E. coli and S. enteritidis. SDS-PAGE patterns of RPMPH revealed light and diffused low molecular weight bands ranging from 8- 25kDa. RPMPH exhibited slightly higher alanine, glycine, and serine content than the native RBPF meat protein. RPMPH showed solubility at a wide range of pH. RPMPH presented the highest emulsification at 2% concentration and foaming at 3% concentration. The water and oil absorption capacities of RPMPH were 2.55 and 1.74mL/ g, respectively. Therefore, RPMPH produced by the probiotic Bacillus (SNGC) strain could be used as a bio-functional ingredient or preservative in food, pharmaceuticals, and cosmetic preparations.