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In previous studies, natural killer lysin (NK-lysin) emerged as a crucial antimicrobial peptide (AMP) discharged by NK cells and CTLs. The sequence of <i>NK-lysin</i> was cloned and discovered in some fishes, but its function remains unclear. In our study, we obtained a copy of <i>NK-lysin</i> from the spleen of the healthy clownfish (<i>Amphiprion ocellaris</i>; <i>AoNK-lysin</i>) through cloning and proceeded to investigate its potential functions and activities. The findings showed that the <i>AoNK-lysin</i> gene’s open reading frame (ORF) had a length of 465 base pairs (bp) and encoded 154 amino acids (aa), which included a saposin B domain and six cysteine residues that are highly conserved, forming three intrachain disulfide bonds to carry out antimicrobial activity. The <i>AoNK-lysin</i> gene was widely present in different tissues, with the skin showing the highest expression, followed by the eye, intestine, and muscle. Additionally, the expression of <i>AoNK-lysin</i> was significantly upregulated in the immune organs (spleen, gill, intestine, and head kidney) of <i>A. ocellaris</i> after being challenged by Singapore group iridovirus (SGIV). Furthermore, a 399 base pair cDNA sequence that encodes the fully developed peptide of AoNK-lysin was successfully inserted into a secretion plasmid called pPIC9K. Subsequently, a significant amount of the recombinant AoNK-lysin protein was efficiently manufactured using the <i>Pichia pastoris</i> expression system. The antibacterial test demonstrated that the AoNK-lysin protein significantly suppressed the growth of various pathogens, particularly <i>Streptococcus agalactiae</i>, <i>Streptococcus iniae</i>, <i>Salmonella typhi</i>, <i>Shigella sonnei</i>, <i>Pseudomonas aeruginosa</i>, and <i>Aeromonas caviae</i>. The minimal inhibitory concentration (MIC) was found to be 7.81 μg/mL. Further analysis of antiviral assays showed all the viral mRNA of SGIV to be significantly reduced after AoNK-lysin protein stimuli in FHM cells. Collectively, these discoveries indicate that AoNK-lysin exhibits features of both direct pathogen-killing abilities and inhibited virus replication.