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<i>Paenibacillus larvae</i> is the causative agent of American foulbrood (AFB), a severe bacterial disease that affects larvae of honeybees. The present study evaluated, in vitro, antimicrobial activity of sixty-one <i>Lactiplantibacillus plantarum</i> strains, against <i>P. larvae</i> ATCC 9545. Five strains (P8, P25, P86, P95 and P100) that showed the greatest antagonism against <i>P. larvae</i> ATCC 9545 were selected for further physiological and biochemical characterizations. In particular, the hydrophobicity, auto-aggregation, exopolysaccharides production, osmotic tolerance, enzymatic activity and carbohydrate assimilation patterns were evaluated. The five <i>L. plantarum</i> selected strains showed suitable physical and biochemical properties for their use as probiotics in the honeybee diet. The selection and availability of new selected bacteria with good functional characteristics and with antagonistic activity against <i>P. larvae</i> opens up interesting perspectives for new biocontrol strategies of diseases such as AFB.