Bacillus amyloliquefaciens strains have been used as biocontrol agents for the suppression of several soilborne plant pathogens. A clearer understanding of the antagonistic mechanisms of action of these bacteria will facilitate their use in the control of plant diseases. Antagonistic substances were isolated from the fermentation broth of B. amyloliquefaciens strain NJN-6 cultures. These compounds were preconcentrated using an XAD-16 column and were purified using reversed-phase high-performance liquid chromatography (RP-HPLC). Fractions were collected from the column and were analyzed, and two homologues of bacillomycin D [molecular weights of 1030 Da (C14) and 1044 Da (C15)] and three homologues of members of the macrolactin family, macrolactin A, 7-O-malonyl macrolactin A, and 7-O-succinyl macrolactin A (molecular weights of 402, 487, and 502 Da, respectively) were identified using HPLC/electrospray ionization mass spectrometry (ESI-MS) analysis. An antagonistic assay showed that bacillomycin D and macrolactin exhibited significant antagonistic effects against Fusarium oxysporum and Ralstonia solanacearum , respectively. A reliable method for the isolation and purification of bacillomycin D and macrolactin from bacterial broth cultures was developed. These data will help elucidate the mechanisms that B. amyloliquefaciens NJN-6 uses for the biocontrol of soilborne plant pathogens.