Chitosan were prepared from cuticle of Lucilia cuprina maggots with two steps; deproteinization and deacetylation. It was characterized with solubility and Fourier Transform Infrared spectroscopy (FT-IR). Chitosan was ball-milled to obtain the chitosan nanoparticles which characterized with dynamic light scattering (DLS) and transmission electron microscope (TEM). Chitosan nanoparticles with degree of deacetylation (DDA) 80.5% were showed antibacterial activities against Klebsiella pneumoniae and Bacillus subtilis. The mode of action of chitosan nanoparticles on the tested bacteria was studied by TEM. Leakage of some cell contents, cell deformation and rupture of cell were observed, therefore, the chitosan nanoparticles were observed to be a powerful antibacterial agent.