Fusarium wilt caused by Fusarium oxysporum has become a serious problem world-wide and relies heavily on chemical fungicides. We selected Pseudomonas sp. NJ134 to develop an effective biocontrol strategy. This strain shows strong antagonistic activity against F. oxysporum. Biochemical analyses of ethyl-acetate extracts of NJ134 culture filtrates showed that 2,4-diacetylphloroglucinol (DAPG) was the major compound inhibiting in vitro growth of F. oxysporum. DAPG production was greatly enhanced in the NJ134 strain by adding mannitol to the growth media, and in vitro antagonistic activity against F. oxysporum increased. Bioformulations developed from growth of NJ134 in sterile bean media with mannitol as the carbon source under plastic bags resulted in effective biocontrol efficacy against Fusarium wilt. The efficacy of the bioformulated product depended on the carbon source and dose. Mannitol amendment in the bean-based formulation showed strong effective biocontrol against tomato Fusarium wilt through increased DAPG levels and a higher cell density compared to that in a glucose-amended formulation. These results suggest that this bioformulated product could be a new effective biocontrol system to control Fusarium wilt in the field.