As part of the ongoing search for novel antimicrobial agents and their use in singular or combined drug therapy, a polypeptide fractions of Mr about 14.500 and 15.00 KDa were isolated from the lymph fluid of two groups of intact and immunized Saudi Arabian honeybees (Apis mellifera) obtained from Al-Qasim and Hail locations around Riyadh during the honey season 2008 following experimental infection with 1.1 x l0⁶ viable Escherichia coli cells(ATCC 25922).  The polypeptide was purified to homogeneity by Reversed Phase High Performance Liquid Chromatography. Antibacterial activity of the isolated polypeptide was evaluated in vitro by an agar well diffusion method for E.  coli strain (ATCC 25922) and Klebsiella pneumoniae strain (ATCC 11678) the major Gram negative pathogens causing urinary tract infections, and Staphylococcus aureus (ATCC 6538) as Gram positive bacteria. A total of ten honey samples collected from different floral areas around Riyadh were also investigated for their antimicrobial activity against one yeast, Candida albicans (ATCC 10231) and four standards bacteriastrains, E. coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), S. aureus (ATCC 6538),  Bacillus subtilis (ATCC 6633) using standard antimicrobial assays.  The isolated polypeptide and the different honey samples revealed comparable marked variations in antimicrobial activities and their sensitivity might be depending on their floral source.