Oilseed rape (OSR) grown in monoculture shows a decline in yield relative to virgin OSR of up to 25%, but the mechanisms responsible are unknown. A long term field experiment of OSR grown in a range of rotations with wheat was used to determine whether shifts in fungal and bacterial populations of the rhizosphere and bulk soil were associated with the development of OSR yield decline. The communities of fungi and bacteria in the rhizosphere and bulk soil from the field experiment were profiled using terminal restriction fragment length polymorphism (TRFLP) and sequencing of cloned internal transcribed spacer regions and 16S rRNA genes, respectively. OSR cropping frequency had no effect on rhizosphere bacterial communities. However, the rhizosphere fungal communities from continuously grown OSR were significantly different to those from other rotations. This was due primarily to an increase in abundance of two fungi which showed 100% and 95% DNA identity to the plant pathogens Olpidium brassicae and Pyrenochaeta lycopersici, respectively. Real-time PCR confirmed that there was significantly more of these fungi in the continuously grown OSR than the other rotations. These two fungi were isolated from the field and used to inoculate OSR and Brassica oleracea grown under controlled conditions in a glasshouse to determine their effect on yield. At high doses, Olpidium brassicae reduced top growth and root biomass in seedlings and reduced branching and subsequent pod and seed production. Pyrenochaeta sp. formed lesions on the roots of seedlings, and at high doses delayed flowering and had a negative impact on seed quantity and quality.