Honeybees harbor a specialized, host-adapted microbial community in their digestive system. This honeybee microbiota lives in a mutualistic relationship with its host and helps the host digest trace sugars found in nectar, degrades polysaccharides found in pollen, stimulates the host’s immune system, excretes antimicrobial compounds which ward off pathogens and promotes host growth and development. There is growing evidence that this microbiota plays an indispensable role in maintaining individual honeybee health and consequently the health as well as productivity of the entire beehive. Metagenomic sequencing has confirmed that Lactic acid bacteria of the genus Lactobacillus are the most abundant members of the native honeybee microbial community and that they have a high degree adaptation to the niche environment of the honeybee gut that distinguishes them from other Lactobacillus species in the same genus. The most prominent adaptations are good tolerance to high osmotic pressure and fructose, as opposed to glucose, as the preferred energy source. The present study attempted to isolate, using MRS medium selective for Lactobacilli, lactic acid bacteria belonging to the genus Lactobacillus from the digestive tract of Carniolan honeybees (Apis mellifera carnica) in their native habitat of western Croatia. In total, 16 microbial isolates were obtained, 6 of which were catalase negative. 3 of these were successfully identified on the genus level as Lactobacilli using MALDI-TOF MS. These 3 bacterial isolates (A6, A10, A15) also showed a good degree of osmotic tolerance with growth observed in sugar syrup solution during 72 hours. The observed characteristics of these 3 isolates correspond to the expected adaptations to honeybee gut environment and the likelihood is high that they are indeed members of honeybee native microbiota.