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Mastitis is an inflammatory condition of the mammary gland, most often caused by bacterial infec-tions, resulting in significant economic losses to the dairy industry. Antimicrobial resistance has been of great concern because of the extensive clinical use of antibiotics. For this reason, the development of new compounds as an alternative treatment to bovine mastitis is needed. Bee venom has been widely used as an oriental treatment for several inflammatory diseases and bacterial infections. The aim of the present study was to evaluate the antimicrobial activity of bee venom on bacteria isolated from bovine mastitis. A total of 107 isolates from bovine mastitic milk samples collected in 2019 and 2020 in Jeonbuk province. All bacterial isolates were tested for susceptibility to bee venom of the honey bee (Apis mellifera ). In order to obtain comprehensive antibacterial activities of the bee venom, we measured the minimal inhibitory concentration (MIC) of the bee venom against bacte-rial strains. Bee venom showed significant inhibition of bacterial growth of Gram-negative bacteria Citrobacter spp., Escherchia coli , Klebsiella spp., Pseudomonas spp., Serratia spp. and Raoultella with MIC values of 96, 81, 72, 230, and 85 μg/mL, respectively, and Gram-positive bacterial Enterococcus spp., Staphylococcus spp. and Streptococcus spp. with MIC values of 29, 21 and 16 μg/mL, respec-tively. The results indicated that the MIC values were different depending on the bacterial strains, and those of Gram-positive bacteria were lower than those of Gram-negative bacteria for bee venom. These findings suggested that bee venom could be an effective antimicrobial treatment for bovine mastitis; however, further research is necessary to evaluate the mechanism underlying the antimicro-bial action, its effectiveness/safety in vivo and effective application for therapeutic use.