AbstractEuropean foulbrood (EFB), a serious disease affecting honey bee larvae, Apis mellifera L. (Hymenoptera: Apidae), is caused by the Gram‐positive bacterium Melissococcus plutonius corrig. (ex White) Bailey & Collins. In this in‐vitro study, we evaluated the inhibitory potential of microbial metabolites produced by lactic acid bacteria (LAB), isolated from the honey bee gut, against M. plutonius. Several LAB species were identified by biochemical tests and 16S rRNA sequencing. Their filtered cell‐free supernatant (CFS) fluids were evaluated individually and in combination against growth and survival of three concentrations of M. plutonius (104–106 colony‐forming units mL−1), for various time intervals (24–96 h), in agar‐well diffusion and broth micro‐dilution assays. We also assessed whether the bioactive metabolites from selected LAB isolates consisted of acids, bacteriocins, and/or hydrogen peroxide, and quantified the presence of short‐chain fatty acids (SCFA; i.e., acetic, butyric, lactic, and propionic acid) by means of high‐performance liquid chromatography. Six LAB isolates inhibited M. plutonius growth: Lactobacillus acidophilus (Moro) Hansen & Mocquot, Lacticaseibacillus rhamnosus (Hansen) Zheng et al., Lactiplantibacillus plantarum (Orla‐Jensen) Zheng et al. (two isolates), Lactobacillus apis Killer et al., and Pediococcus acidilactici Lindner. The inhibitory effects on M. plutonius were larger at the lowest than at the highest M. plutonius concentration, and P. acidilactici showed the strongest antibacterial activity. Exposure to a mixture of CFS fluids of the four most potent isolates could reduce M. plutonius survival to 0% within 96 h. The antagonistic activity in the CFS of L. rhamnosus and L. apis appeared to be related to the presence of acids, whereas the antagonistic activity in L. plantarum and P. acidilactici appeared to be related to the presence of bacteriocins or bacteriocin‐like inhibitory substances (BLIS). Hydrogen peroxide did not seem to play a role. Total SCFA levels were highest in L. rhamnosus CFS fluid. These results can serve as a basis for in vivo studies on the use of LAB isolates as potential biocontrol agents against EFB in honey bees.