The impact of different concentrations of red-bellied pacu fish meat protein hydrolysate (RPMPH) on the physicochemical, sensory, and storage properties of cookies was studied. RPMPH was prepared from red-bellied pacu fish meat by probiotic Bacillus fermentation for 48 h. RPMPH showed 87.48% protein content with DPPH scavenging activity (71.22%), ABTS scavenging activity (90.05%), ferric reducing antioxidant power (278.13 μM TE/g), and Fe2+ chelating activity (65.46%). Cookies incorporated with RPMPH displayed a higher protein content (11.76–14.40%) and in vitro protein digestibility (65.21–96.0%) compared to control (without RPMPH) cookies (P < 0.05). The total dietary fiber content of all cookies ranged from 1.74 to 2.63 g/100 g sample. The control-cookies had a higher diameter (7.0 mm) and spread ratio (8.37%) compared to cookies incorporated with RPMPH (P < 0.05). The cookies incorporated with RPMPH exhibited a slightly higher water absorption index (6.79–7.51%), water solubility index (2.74–2.88%), and hardness (41.36–49.48 N) than the control-cookies. Color of cookies, influenced by incorporation of RPMPH. Further, the cookies incorporated with RPMPH showed slightly higher moisture and water activity compared to control-cookies throughout the storage (P < 0.05). Conversely, cookies incorporated with RPMPH displayed lower free fatty acids, peroxide values, and thiobarbituric acid substances than control-cookies (P < 0.05) throughout the storage period. The cookies with 8% RPMPH exhibited a higher score for overall acceptability and presented a better amount of essential amino acids. The present study suggested that 8% RPMPH can be incorporated into the cookie formulation, enhancing the nutritional profile and storage stability.