Sardinelle protein hydrolysate (SPH), prepared by treatment with Bacillus subtilis A26 proteases, was found to exhibit antibacterial, antioxidant and ACE-inhibitory activities. SPH, with a degree of hydrolysis of 4%, was fractionated by size exclusion chromatography on a Sephadex G-25 into five major fractions (F1-F5). F2, which exhibited the highest antibacterial and ACE-inhibitory activities, and F4, which exhibited the highest antibacterial and antioxidant activities, were further fractionated by reverse phase-high performance liquid chromatography (RP-HPLC) and then analysed using nano-ESI-LC-MS/MS to identify the sequences of peptides. Eight peptides were identified in the sub-fraction F2-A, nine peptides in the sub-fraction F4-B, and 45 peptides in F4-C. Identified peptides were found to share sequences with previously described bioactive peptides based on Biopep database. The results of this study suggest that SPH is a good source of natural bioactive peptides. Hence, it can be used as a potential ingredient in nutraceutical field.