Antimicrobial peptides constitute an important component of the innate immune system. Teleost fish represent a potentially fruitful resource for novel antimicrobial peptide discovery since these organisms rely significantly on their innate immune systems to combat the constant threat of infections in the aquatic environment. In the present study, we isolated three antimicrobial peptides-like transcripts from tilapia (Oreochromis niloticus) gills based on EST reported sequences. These peptides were named oreochromicins (Oreoch-1, Oreoch-2 and Oreoch-3). The cDNA sequences for these putative AMPs encode three pre-pro-peptides with the highest similarity with the members of the piscidin family from teleost fish. The predicted three pre-pro-peptides consist of a signal peptide, a highly cationic mature peptide of 23, 25, and 32 amino acids, respectively and a carboxy terminal pro-domain. The synthetic peptides displayed a broad-spectrum of antimicrobial activity against Gram-negative, Gram-positive bacteria and fungi. These peptides are constitutively expressed in the brain, heart, head kidney, spleen and gut. Additionally, their binding properties to lipopolysaccharide and cytotoxic activity in mammalian and fish cells were assayed.