BackgroundAmerican foulbrood (AFB) disease caused byPaenibacillus larvaeis dangerous, and threatens beekeeping. The eco-friendly treatment method using probiotics is expected to be the prospective method for controlling this pathogen in honey bees. Therefore, this study investigated the bacterial species that have antimicrobial activity againstP. larvae.ResultsOverall, 67 strains of the gut microbiome were isolated and identified in three phyla; the isolates had the following prevalence rates: Firmicutes 41/67 (61.19%), Actinobacteria 24/67 (35.82%), and Proteobacteria 2/67 (2.99%). Antimicrobial properties againstP. larvaeon agar plates were seen in 20 isolates of the genusLactobacillus, Firmicutes phylum. Six representative strains from each species (L. apisHSY8_B25,L. panisapiumPKH2_L3,L. melliventrisHSY3_B5,L. kimbladiiAHS3_B36,L. kullabergensisOMG2_B25, andL. mellisOMG2_B33) with the largest inhibition zones on agar plates were selected for in vitro larvae rearing challenges. The results showed that three isolates (L. apisHSY8_B25,L. panisapiumPKH2_L3, andL. melliventrisHSY3_B5) had the potential to be probiotic candidates with the properties of safety to larvae, inhibition againstP. larvaein infected larvae, and high adhesion ability.ConclusionsOverall, 20 strains of the genusLactobacilluswith antimicrobial properties againstP. larvaewere identified in this study. Three representative strains from different species (L. apisHSY8_B25,L. panisapiumPKH2_L3, andL. melliventrisHSY3_B5) were evaluated to be potential probiotic candidates and were selected for probiotic development for the prevention of AFB. Importantly, the speciesL. panisapiumisolated from larvae was identified with antimicrobial activity for the first time in this study.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-023-02902-0.