We investigated the molecular characterization of a Tilapia by-product hydrolysate to asses its nutritional potential in aquafeed. The proximate composition and amino acid composition showed that the hydrolysate had high protein content and contained all the essential amino acids required for carnivorous fish nutrition. The molecular weight profile determined by High Performance Size Exclusion Chromatography (HPSEC) indicated that the hydrolysate was mainly composed of polypeptides (46.9%) and oligopeptides (35.8%). We also characterized the peptide fraction by nLC-ESI-MS/MS and OFFGEL-nLC-MALDI-TOF/TOF. We identified 1374 peptides, and highlighted a high diversity of oligopeptides and polyp eptides. Finally, the Tilapia by-product hydrolysate exhibited antimicrobial activity against Yersinia ruckeri and to a lesser extent against Edwardsiella tarda and Bacillus megaterium. These preliminary results suggest the strong nutritional and functional potential of Tilapia by-product hydrolysates. (C) 2014 Elsevier Ltd. All rights reserved.