Bacillus species produce a wide range of secondary metabolites that display a broad spectrum of antimicrobial activity and various molecular structures. In this study, cyclic lipopeptides were produced by Bacillus amyloliquefaciens strain BC32-1 that was isolated from red clay soil in the Jeonnam province of South Korea. The BC32-1 strain is one of effective microbes for the biocontrol of plant diseases caused by soil-borne, foliar, and post-harvest fungal pathogens. The BC32-1 strain strongly inhibited mycelial growth of plant pathogenic fungi Fusarium oxysporum f.sp. radicis-lycopersici which infects a broad range of host species, causing wilt or rot disease in significant agricultural plants. Several lipopeptides were purified from culture media by organic solvent extraction and reversed-phase high performance liquid chromatography (RP-HPLC). The purified lipopeptides contained iturin-, surfactin-, and fengycin-type lipopeptides, as confirmed by liquid chromatography-mass/mass spectrometry (LC-MS/MS) analyses. Among the lipopeptides, C17-fengycin B showed higher antifungal activity and the molecular structure was further characterized using UV, Fourier transform-infrared spectroscopy (FT-IR), LC-MS/MS, and NMR spectroscopy. The fengycin was highly produced up to 15 mg/mL at 37°C, whereas the production of the fengycin was very low at 25°C. The purified fengycin inhibited mycelial growth of F. oxysporum with a minimal inhibitory concentration (MIC) of 50 mg/mL. This study suggests that C17-fengycin B is an important antifungal component produced by the BC32-1 strain that could be used as an environmentally friendly agent to control the phytopathogenic F. oxysporum. In addition, we are synthesizing various lipopeptide analogs using solid-phase peptide synthesis in combination with structure-based design.