Honeybees are vulnerable to many diseases, such as European foulbrood, chalkbrood, sacbrood, and American foulbrood (AFB). Caused by Paenibacillus larvae, AFB can cause immense damage to the bee industry. Chemicals and antibiotics have long been used to treat these diseases, resulting in pathogen resistance. As an alternative, antagonistic effect of gut bacteria has been focused on. The culture supernatant (CS) from recently isolated gut bacteria from Thai Apis cerena indica (Bacillus sp., Pantoea sp., Azotobacter sp., Klebsiella sp., and Lactobacillus sp.) were screened for their growth inhibitory activity against P. larvae strains 01 and 02 in an agar well diffusion assay. The CS from Azotobacter sp. had the highest growth inhibition against P. larvae strain 02, while the CS from Bacillus sp., Lactobacillus sp., Klebsiella sp., Azotobacter sp. were all active against both strains. This activity was thermotolerant to 90 ºC for 1 h, but 121 ºC nor to proteinase K and RNase A treatment. Furthermore, the CSs from Bacillus sp. and Klebsiella sp. at pH 3, Pantoea sp. at pH 5, Lactobacillus sp. at pH 8 and 10, and Klebsiella sp. at pH 10 showed the highest inhibitory effect against P. larvae strains 01 and 02. However, the CS inhibitory activity at pH 8–10 was less than that at pH 5 (the pH in the bee stomach). Hence, gut bacteria that can potentially inhibit P. larvae growth were obtained from healthy honeybees.