Abstract. Magdalena S, Anggelia, Yogiara. 2020. Characterization of antibacterial activity produced by Bacillus spp. isolated from honey and bee-associated products against foodborne pathogens. Bioteknologi 17: 51-59. Four potential isolates (Bacillus velezensis Y12, Bacillus amyloliquefaciens Y21, Bacillus amyloliquefaciens Y23, and Bacillus velezensis Y33) isolated from honey, propolis, and bee pollen from West Java, Indonesia showed antifungal and enzymatic activities. This study aimed to assay antibacterial activity against foodborne pathogens, determine the optimum condition for antibacterial compounds production, observe the effect of thermal and pH treatment on the antibacterial compounds, and detect the presence of antibacterial peptide biosynthesis gene. In this research, bacterial isolates showed antibacterial activity against Bacillus cereus ATCC 14579, Salmonella enterica 51741, and Salmonella Typhimurium ATCC 14028. The growth condition for antibacterial production of isolate B. velezensis Y12 was at the range of 25-60°C and pH 7-9. Meanwhile, the other three isolates showed the same pattern, also at the range of 37-60 °C. Antibacterial compounds against B. cereus were found heat stable at 4-90°C and active over pH 3-9. Cultivation of the isolates in BHIB and TSB did not significantly increase the antibacterial activity against all pathogens as compared to an LB medium. On the contrary, the antibacterial activity against S. typhimurium of the isolates cultivated in optimized medium two (yeast extract 32.5 g/L, glucose 33.4 g/L, MnSO4 0.042 g/L, CaCl2 0.031 g/L, KH2PO4 0.5 g/L, K2HPO4 0.5 g/L, (NH4)2SO4 1.0 g/L, and MgSO4 4.0 g/L) was significantly increased. Furthermore, seven antibacterial peptide biosynthesis genes primers were amplified from the genomic DNA of isolates B. velezensis Y12 and B. velezensis Y33 by PCR analysis. These genes were fenD, srfAA, bacA, bmyB, ituC, ituD, and bmyD. Meanwhile, there was no presence of ituC gene from the other isolates. This result suggests that isolate B. velezensisY12 might be the most potential isolate for antibacterial compounds production.