The objective of this study was to isolate and characterize bacterial strains associated with the gut of the hybrid Carniolan honey bee, Apis mellifera carnica, and to determine their in vitro and in vivo potential against Ascosphaera apis, the causal organism of chalkbrood disease, with the purpose of exploring feasible biological control. Six bacterial strains were isolated from healthy worker honey bees by culture-dependent methods. Six fungal strains (A3, A4, A7, A8, A9, and A15) of A. apis were isolated from larvae suffering from chalkbrood disease on Yeast-Glucose-Starch agar (YGPSA) medium. All bacteria were identified by a combination of morphology, Gram stain, and 16S rRNA sequence analysis, and fungal strains were identified by morphology and 5.8S rRNA. In vitro and in vivo inhibition assays were carried out to determine the ability of bacterial isolates to inhibit A. apis, the causal agent of chalkbrood disease. The analysis of 16S rRNA sequences revealed that four bacterial strains (B2, B4, B10, and B100) belong to Bacillus subtilis species, and two strains (P1 and P5) belong to Pseudomonas fluorescence. Significant differences in antagonistic activity of all bacterial strains were observed. B. subtilis isolate B2 showed the highest antagonistic activity, as measured by the inhibition zone against A. apis, followed by the P1 strain of P. fluorescence. SEM analysis also supports the antagonistic activity of these bacteria against A. apis. This study provides a theoretical basis for biological control of honey bee chalkbrood disease.