This study investigated the antimicrobial activity of body and ovary homogenates of emerging queens of the honeybee Apis mellifera L. Queens'.  Fourth-instar larvae were challenged with a sublethal dose of Paenibacillus larvae to investigate the effect against multidrug-resistant bacteria (Escherichia coli 1 and 2and Klebsiella pneumoniae) and the causative agent of American Foulbrood (Paenibacillus l. l). The agar disk diffusion test was used. Against the selected pathogenic bacteria, both the body and the ovary homogenates showed antibacterial activity. A highly significant change in the antibacterial activities of total body homogenate was detected for treated queens against E. coli 2 as well as K. pneumoniae (0.65 ± 0.02 and 0.65 ± 0.01, respectively) compared with healthy queens (0.45 ± 0.02 and 0.45 ± 0.02, respectively). On the other hand, the ovary extracts of challenged queens showed highly significant increases in antibacterial activity against P. l. larvae, E. coli 1, and K. pneumonia (0.92 ± 0.05, 0.85 ± 0.4, and 0.67 ± 0.0004, respectively) compared with the healthy queens (0.55 ± 0.02, 0.65 ± 0.23, and 0.49 ± 0.001, respectively). These findings could provide us with essential points on the development and production of new antibiotic agents against bacterial diseases