In our previous study, spiny head croaker (Collichthys lucidus) by-products fermentation (SBF) products demonstrated plant growth promoting effects. In this study, the antimicrobial activities of SBF against plant pathogenic fungi and food-borne bacteria were evaluated. An active antifungal fraction SBF-3-1 was isolated from SBF using size exclusion chromatography and high performance liquid chromatography. Seven short peptides, i.e., LDEGW, DSFDFK, LDGW, FPDL, YNLDFK, FDGF and LDFE, plus one 22 residues peptide TFNTPAMYVAIQAVLSLYASGR were identified in SBF-3-1 by liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI-Q-TOF-MS/MS). The peptide TFNTPAMYVAIQAVLSLYASGR (named as SBF-3-1p), a potential antimicrobial peptide predicted by the antimicrobial peptide database (APD, http://aps.unmc.edu/AP/main.php), was further characterized. The prediction showed that the generation of SBF-3-1p could be associated with the action of trypsin and glutamyl endopeptidase, two proteases with specific cleavage sites on the carboxyl side of arginine (R) and lysine (K), and on the carboxyl side of Glu (E), respectively. According to the structural modeling, SBF-3-1p contained one alpha-helix and two loop regions. The synthesized SBF-3-lp exhibited strong activity against plant pathogenic fungus Pestalotiopsis sp. Therefore, SBF should be a good source of antifungal peptide, and could be used as natural fungicides in agriculture.