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The cDNa sequence of Bestrophin‐1 (BEST‐1) was identified from a previously constructed transcriptome data set of freshwater prawn Macrobrachium rosenbergii(Mr). Basal and temporal gene expression analysis of MrBEST‐1 showed its antimicrobial immune effectiveness during viral and bacterial infections. The protein sequence encoded by cDNA of MrBEST‐1 was examined and a short antimicrobial molecule, named WP17 was identified using a bioinformatics tool. Further, the antibacterial ability of the identified WP17 peptide was evaluated against a number of bacterial strains, in which the peptide showed potential bactericidal activity against Micrococcus luteus(MTCC 6164), Staphylococcus aureus(ATCC 9144), Escherichia coli(ATCC 9637), Klebsiella pneumonia(CI 7376) and Bacillus subtilis(ATCC 6051). Based on the results, further assays focused on M. luteusMTCC 6164. The mode of action of MrWP17 on M. luteusMTCC 6164 was analyzed using FACS and FESEM. Toxicity analysis suggested that WP17 impaired the viability of cells in murine melanoma cells (B16F10); however, no cytotoxicity was observed against kidney embryonic cells (HEK293), even at higher concentrations. Similarly, the gene expression analysis of WP17 peptide treated murine cells elicited an extrinsic apoptotic pathway. In the present study, we have demonstrated the involvement of MrBEST‐1 in immune mechanisms through its short peptide molecule that has potential antimicrobial activity.