This study aimed to isolate lactic acid bacteria from gastrointestinal tract of honey bees (Apis mellifera) and assess the probiotic potential of isolates, and their effect on some performance parameters of colonies. Therefor, 10 colonies from the apiary with one hundred bee colonies were randomly selected and used for sampling. Twenty worker bees were taken from each hive and after being transferred to the laboratory under complete comfort and ventilation conditions, their gastrointestinal tract was completely isolated under carbon dioxide flushing and after culture in MRS medium different colonies were selected. Liquid-solid periodic cultures were repeated up to 8 steps to achieve pure cultures. Finally, gram-positive, catalase and hemolysis negative samples were isolated and identified based on phylogenetic analysis. These bacteria were registered in the Gene bank (NCBI) with accession number of MW376601, MW376904, MW376896 and MW405566, respectively. In vivo studies showed the positive effects of treatment with isolates by spraying on frames and bacterial culture mixed in syrup (sugar - water) on spawning, production and hygienic behavior of bees. Comparison of treatments with Duncan's multiple range test (p≤ 5%) showed that all experimental treatments in quantitative population factors and spawning and quality were significantly different from the control treatment. However, the control treatment had a significant difference in honey reserves due to low spawning compared to experimental treatments and less nectar consumption.