A three-phased approach was used to identify fungi that suppress the plant-parasitic nematode, Heterodera schachtii, in a southern Californian soil. The first phase led to the identification of fungal rRNA genes correlating with the suppressiveness. Soils with various levels of suppressiveness were planted with mustard greens and infested with H. schachtii juveniles. After two nematode generations, the abundance of Dactylella oviparasitica rRNA genes positively correlated with high levels of suppressiveness while the abundance of Fusarium oxysporum rRNA genes positively correlated with minimal to moderate levels of suppressiveness. In phase 2, quantitative PCR analyses corroborated the population trends identified in phase 1. In phase 3, both fungi were added to fumigation-induced non-suppressive soil, planted with Swiss chard and infested with H. schachtii juveniles. After two nematode generations, D. oviparasitica applications reduced the population densities of H. schachtii eggs and juveniles to those in the suppressive soil and H. schachtii cysts to levels lower than in the suppressive soil. While F. oxysporum applications consistently reduced H. schachtii population densities compared to the non-suppressive soil, these levels were not significantly different. These results suggest that D. oviparasitica plays a major role in the H. schachtii suppressiveness exhibited by this soil. Subsequent experimentation demonstrated that D. oviparasitica applications could suppress H. schachtii populations in five different soil types. D. oviparasitica applications were also able to produce H. schachtii suppressiveness in field microplot experiments. Overall, these data suggest that this general approach could be useful for investigations of other suppressive soils as well as for studies aimed at identifying microorganisms involved in other in situ functions.