The pathogenicity of Cylindrocarpon didymum towards roots of subterranean clover (Trifolium subterraneum) at four inoculum rates (0.5, 1.0, 2.0, 5.0% w/w) under three temperature regimes (15/10, 18/13, 21/16°C) and the ability of C. didymum to produce the mycotoxin brefeldin A in vitro was determined. C. didymum infected and damaged tap and lateral roots of subterranean clover and reduced plant dry weights, particularly when sufficiently high levels of inoculum were present. Most disease occurred at the cooler temperature regime of 15/10°C, a temperature regime that would closely approximate field soil temperatures in the lower south-west of Western Australia during the winter months. C. didymum produced brefeldin A in vitro and the frequently observed, stunted appearance of affected tap and lateral roots was possibly as a consequence of production of brefeldin A in the root tissue by this pathogen. This is the first published report of C. didymum as a root pathogen of subterranean clover.