Tomato vine decline (TVD) in south-western Ontario is a disease with symptoms that include premature plant senescence, root browning and rotting, and fruit yield reduction. Soilborne fungi cause similar vine decline symptoms in melons and potatoes. To identify which potential pathogens might be associated with TVD, we undertook both a DNA and culture based survey of the fungi found in roots and rhizospheres of field and lab grown plants. DNA finger printing revealed that roots are colonized by significantly different fungal populations than those found in rhizospheres or the soil in which they are grown. The fungal species associated with TVD resistant 'Beaufort' and 'RST-04–105-T' rootstocks were also consistently different from those in the TVD susceptible commercial variety 'Heinz 2401ʹ. Mycobiomes in 'Heinz 2401ʹ roots and rhizospheres changed significantly when plants were grown in sterile sand, TVD infested soil, or in the same soil that had been amended with molasses. Internal transcribed spacer (ITS) region sequences derived from PCR product clones or fungal cultures isolated from roots identified 53 different fungal species. Annotated DNA fingerprints showed that roots of all plants grown in soil contained Verticillium dahliae, a destructive vascular pathogen of tomatoes, while only susceptible 'Heinz 2401ʹ roots were infected with the obligate parasite Olpidium virulentus. While these two pathogens may both be reducing tomato yields in Ontario, it appears that existing tomato rootstock varieties may be resistant to infection by O. virulentus. This poorly characterized soil-transmitted Chrytidiomycete may be contributing to TVD.