Biological soil disinfestation (BSD) is one of the methods for soil disinfestation recently developed and consists of organic amendment, irrigation, and covering the soil surface with plastic film. BSD trials with artificially infested soils effectively killed Fusarium oxysporum f. sp. lycopersici and Ralstonia solanacearum. F. oxysporum f. sp. lycopersici was not detected 9 days after treatment. Application of BSD to R. solanacearum-infested soil decreased disease severity 4 weeks after transplantation. Reduction in soil pH and Eh was observed in the BSD-treated soil. HPLC analysis revealed the accumulation of acetic acid and butyric acid in the soil and the disinfestation effect of BSD was almost reproduced by mixing these organic acids with the infested soil. Volatile compound(s) evolved in the BSD-treated soil was also suggested to contribute to disinfestation. PCR-DGGE (PCR-denaturing gradient gel electrophoresis) analysis revealed the increase in the abundance of several bacteria in the BSD-treated soil, and two of them had 100% similarity with potential organic acid producers, Clostridium sp. and Enterobacter sp.