A non-mycoparasitic restriction enzyme-mediated DNA integration (REMI) mutant of Coniothyrium minitans (R2427) contains two tandem plasmid copies integrated towards the 3' end of an ORF. The predicted polypeptide (845 aa) exhibits high similarity with DNA-helicase proteins from other filamentous fungi and yeasts that play a role in mitochondrial DNA maintenance and repair. Disruption of the C. minitans PIF1 DNA helicase gene results in altered morphology, reduced growth rates and a concomitant loss in ability to mycoparasitize sclerotia of Sclerotinia sclerotiorum. In infection bioassays, R2427 exhibited sparse mycelial growth on the surface of live sclerotia, but no mycelia were detected inside the sclerotia. Conversely, R2427 readily colonized autoclaved sclerotia. Complementation of the mutant with wild-type PIF1 restored normal mycelial growth and mycoparasitic capability, confirming a functional role in the host-pathogen interaction. The C. minitans PIF1 DNA helicase may maintain mitochondrial stability in response to reactive oxygen species, either produced endogenously within the mycoparasite, or exogenously from the sclerotial host.