Antimicrobial peptide crustin was isolated and purified from Penaeus semisulcatus using Sephadox G-100 column gel filtration chromatography. P. semisulcatus crustins was observed as a single band with 14 kDa of molecular weight on SDS-PAGE and the retention time of 46 min in RP-HPLC. Circular dichroism spectra of P. semisulcatus crustin showed alpha helices in its secondary structure followed by random coils. Crystalline nature and functional groups arrangement were investigated by X-Ray Diffraction (XRD) and Fourier Transform Infra-Red spectroscopy (FTIR). P. semisulcatus crustin showed the effective antibacterial activity against Gram positive strains B. thuringienisis (4 μg/ml) and B. pumilis (6 μg/ml) when compare to Gram negative strains. Biofilm Inhibitory Concentration (BIC) were determined for these strains and percentage of biofilm inhibition was confirmed and visualized through in sit microscopic analysis. Hence, we reported the effect of crustin on biofilm inhibition and eradication at low concentrations by using crystal violet staining and confocal microscopic observations. In addition, haemolytic activity of this purified crustin also analysed using human RBCs. The results of this study, suggests that this bio peptide crustin is a potential and promising therapeutic agent to treat drug resistant bacteria and biofilm-related infections.