Biological soil disinfestation (BSD) or reductive soil disinfestation (RSD) is an environmental biotechnology to eliminate soil-borne plant pathogens based on functions of indigenous microbes. BSD treatments using different types of organic materials have been reported to effectively control a wide range of plant pathogens. Various studies have shown that development of reducing or anoxic conditions in soil is the most important aspect for effective BSD treatments. Substances such as organic acids, FeS, or phenolic compounds generated in the treated soil have been suggested to contribute to inactivation of pathogens. Additionally, anaerobic bacteria grown in the BSD-treated soil may produce and release enzymes with anti-pathogenic activities in soil. Clone library analyses as well as a next-generation sequence analysis based on 16S rRNA genes have revealed prosperity of obligate anaerobic bacteria from the class Clostridia in differently treated BSD soils. Two anaerobic bacterial strains isolated from BSD-treated soil samples and identified as Clostridium beijerinckii were found to decompose major cell wall polysaccharides of ascomycetous fungi, chitosan and beta-1,3-glucan. C. beijerinckii is a species most frequently detected in the clone library analyses for various BSD-treated soils as a closely related species. The two anaerobic isolates severely degraded mycelial cells of the Fusarium pathogen of spinach wilt disease during anaerobic co-incubation of each isolate and the Fusarium pathogen. These reports suggest that antifungal enzymes produced by predominant anaerobic bacteria grown in the BSD-treated soil play important roles to control soil-borne fungal pathogens. Further studies using different bacterial isolates from BSD-treated soils are expected to know their anti-pathogenic abilities.