The objective of this study was to determine the antioxidant activity and possible mode of action of partially purified peptides derived from threadfin bream surimi byproduct. The frame, bone and skin (FBS) byproduct, which was obtained from a deboning process, was hydrolyzed by Virgibacillus sp. SK33 proteinase and fractionated using anion exchange and size exclusion chromatography. Three fractions, i.e., B1, B2 and B3, were obtained, and the amino acid sequences of the peptides in all 3 fractions were determined using LC-MS/MS. Fractions B2 and B3 contained higher amounts of Trp, Met, Cys and Tyr residues than fraction B1. Fraction B3 exhibited high ABTS radical scavenging activity and ferric reducing antioxidant power (FRAP), while metal chelation and hydroxyl radical scavenging ability were principle modes of action of peptides in fraction B2 and B3. In addition, fraction B1 and a synthetic peptide selected from the pooled de novo peptides of fraction B3, FLGSFLYEYSR, had a cellular radical scavenging effect when HepG2 cells were treated with H2O2.