Biosurfactants or microbial surfactants are structurally diverse and contribute to reducing surface and interfacial tension. Apart from being isolated from municipal waste and oil contaminated soil, biosurfactant-producing Bacillus spp. can also be found in fermented foods. Bacillus subtilis produce surfactant, which exhibits antibacterial activity against various pathogenic bacteria. In this study, surfactant-producing Bacillus were isolated from traditional fermented food namely; budu (fish sauce), cincalok (fermented small shrimps), tempeh (fermented soybeans) and tapai ubi kayu (fermented cassava). Identification of surfactin-producing bacteria was conducted using 16S rRNA which indicated that surfactin-producing strains isolated from budu and tempeh are Bacillus subtilis while strains from cincalok and tapai ubi kayu are Bacillus amyloliquefaciens with a sequence similarity of 99%. Characterization of surfactin was carried out by High-Performance Liquid Chromatography (HPLC) and Liquid Chromatography-Mass Spectrometry (LCMS). HLPC analyzes identified major peaks contributing to the presence of surfactin. LCMS analysis detected specific mass to charge ratio (m/z) which contributed to the presence of maximum thirteen surfactin isoforms. Surfactin produced by the isolated strains also exhibited antibacterial activity toward pathogenic bacteria, namely Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumoniae, Salmonella Typhimurium, Serratia marcescens and Klebsiella pneumoniae. All isolated strains from the Malaysian fermented food types investigated in this study were able to produce surfactin. In addition, antibacterial screening via agar well diffusion, measurements of MBC and MIC show surfactin produced by the isolated strains have antibacterial activities toward certain Gram-positive and Gram-negative bacteria tested in this study.