Abstract: Background and Objective: Lactic acid bacteria are generally recognized as safe that could be beneficial for several uses in food industry to get their health benefits. The present study was focused on the isolation and identification of some new lactic acid bacteria that might be naturally occurred in the honeybees stomach and tried to explore their benefits. Materials and Methods: Twenty five isolates of lactic acid bacteria were isolated from the stomach of three different types of Egyptian bees (Apis mellifera lamarckii ), Carniolan bees (A.m. carnica) and hybrid Carniolan bees. Identification of isolates was carried out based on phenotypical tests and carbohydrate assimilation using API50 CHL and 16S rDNA sequencing. Results: In the present study, the results emphasized Lactobacillus plantarum to be the predominant species (62.5%), other strains were identified as Pediococcus pentosaceus (12.5%), Lactobacillus pentosus (12.5%) and Lactobacillus sakei (12.5%). Eight of 25 isolates showed a potential antibacterial activity especially against Salmonella senftenberg strain. The novel isolates (HBMSS1, HBMSS3, HBMSS4, HBMSS5, HBMSS6 and HBMSS8) showed a significant antimicrobial activity against C. botulinum, E. coli, S. Senftenberg and S. epidermidis as food borne pathogens and P. larvae and M. plutonius as honeybee pathogens. Conclusion: These promising findings might be beneficial for discovering novel preservatives in food industry and substitution of antibiotic drugs used in the treatment of honeybees’ infection.