Stonebrood is a virulent disease afflicting honey bee colonies. Bacterial strains present in the gut of the Egyptian honeybee, Apis mellifera were isolated, identified and characterized. Moreover, its inhibitory effect against the etiological agent of Stonebrood disease was determined according to their antifungal activity. Both Gram-positive and Gram-negative bacteria were observed in all seasons. The spring season was found to possesses the highest abundance and variety of these bacteria. A total of 93 bacterial isolates from honeybee gut microbiota were identified by Vitek Ms method elucidating that bacterial populations in the gut of the Egyptian honeybee consist of, Firmicutes phyla (75.2%), Bacteroidetes (3.22%), Alpha-Proteobacteria (1.07%) and Gamma-Proteobacteria (20.4%). In vitro antifungal activity was carried out to determine the ability of Cell-Free Supernatants (CFS) to inhibit two Aspergillus sp., the causal agent of the Stonebrood disease. Out of 93 isolates, three of them exhibited potent antifungal activity against Aspergillus niger and A. flavus. Most antagonistic CFS identifications were performed by 16S rRNA gene sequence analysis revealing that bacterial isolates belong to Sphingomonas paucimobilis, Staphylococcus aureus, Pseudomonas aeruginosa and Lactobacillus kunkeei. Bacterial isolates obtained in this study may be potential candidates as biological control agents against Aspergillus sp. [ABSTRACT FROM AUTHOR]