The aim of the present study was to extract chitosan from corpses of the naturally died honeybee, prepare chitosan nanoparticles (B- CS- NPs) for loading the honeybee, Apis mellifera venom (BV) and evaluate their antimicrobial potential. Chitin was extracted from the cuticle of corpses of naturally died honeybees following 4 steps; de-waxing, demineralization, deproteinization and discoloration. Chitosan was obtained by deacetylation of chitin and characterized using the Fourier transform infrared (FTIR) and X-ray diffraction. Honeybee chitosan nanoparticles (B-CS-NPs) were prepared by ionic gelation method using TPP in acidic medium. Empty nanoparticles (B-CS-NPs) and bee venom loaded nanoparticles (BV loaded NPs) were characterized. Hydrodynamic size and zeta potential of B- CS NPs were 74.2 nm, and 51.1 mV, while those of bee venom-loaded (BV loaded NPs) were 110.5 nm, 49.0 mv, respectively. The loading capacity (LC) and encapsulation efficiency (EE) were 86.5 % and 91.3 %, respectively, at bee venom concentration of 600 µg / ml. The antimicrobial activity of empty and BV loaded nanoparticles was studied using different strains of human pathogenic bacteria and fungi. Compared to empty nanoparticles, BV loaded NPs exhibited potent antimicrobial activity against the studied strains except in Aspergillus flavus fungus, which seemed to be resistant.