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Lactic acid bacteria (LAB) are an important group of honeybee gut microbiota. These bacteria are involved in food digestion, stimulate the immune system, and may antagonize undesirable microorganisms in the gastrointestinal tract. <i>Lactobacillus kunkeei</i> is a fructophilic lactic acid bacterium (FLAB) most frequently found in the gastrointestinal tracts of honeybees. <i>Ascosphaera apis</i> is an important pathogenic fungus of honeybee larvae; it can colonize the intestine, especially in conditions of nutritional or environmental stress that cause microbial dysbiosis. In this work, some functional properties of nine selected <i>L. kunkeei</i> strains were evaluated. The study focused on the antifungal activity of these strains against <i>A. apis</i> DSM 3116, using different matrices: cell lysate, broth culture, cell-free supernatant, and cell pellet. The cell lysate showed the highest antifungal activity. Moreover, the strains were shown to possess good cell-surface properties (hydrophobicity, auto-aggregation, and biofilm production) and a good resistance to high sugar concentrations. These <i>L. kunkeei</i> strains were demonstrated to be functional for use in “probiotic syrup”, useful to restore the symbiotic communities of the intestine in case of dysbiosis and to exert a prophylactic action against <i>A. apis.</i>