As part of the ongoing search for novel antimicrobial agents and their use in singular or combined drug therapy, peptide fractions of molecular weights about 14.500 and 15.00kDa were isolated from the hemolymph of wild (Apis florae) and carniolan (Apis carnica) bees of Saudi Arabia obtained from different regions in Riyadh (variable plant sources) during the honey seasons (spring and summer) 2008–2009. Following experimental infection with 1.1×l06 viable Escherichia coli cells (ATCC 25922), the antimicrobial peptides were purified to homogeneity by reversed-phase high performance liquid chromatography. Antibacterial activity of the isolated peptide 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 10 honey samples collected from bee hives selected arbitrary at different floral areas of south Riyadh were also investigated for their antimicrobial activities against the yeast, Candida albicans (ATCC 10231) and four standard bacteria strains, E. coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), S. aureus (ATCC 6538) and Bacillus subtilis (ATCC 6633) using standard antimicrobial assays. The isolated antibacterial peptides and the different honey samples revealed comparable marked variations in antimicrobial activities and their sensitivity might be depending on their variable floral sources.