Gram-positive antagonistic bacilli were isolated from agricultural soils for possible use in biocontrol of plant pathogenic fungi, Fusarium oxysporum, Rhizoctonia solani, and/or Pythium ultimum. Among the 65 antagonistic Gram-positive soil isolates, 22 strains were identified as Bacillus species by 16S rDNA sequence analyses. Four strains, including DF14, especially exhibited multiple antagonistic properties against the three damping-off fungi. Genotypic properties of the Bacillus isolates were characterized by rapid molecular fingerprinting methods using repetitive extragenic palindromic-PCR (REP-PCR), ribosomal intergenic spacer-length polymorphisms (RIS-LP), 16S rDNA PCR-restriction fragment length polymorphisms (PCR-RFLP), and strain-specific PCR assays. The results indicated that the REP-PCR method was more valuable than the RIS-LP and 16S rDNA PCR-RFLP analyses as a rapid and reliable approach for bacilli typing and identification. The use of strain-specific primers designed based on 16S rDNA sequence comparisons enabled it to be possible to selectively detect a strain, DF14, which is being used as a biocontrol agent against damping-off fungi.