Hymenoscyphus pseudoalbidus is the causal agent of ash dieback, a disease that is presently endangering Fraxinus spp. throughout most of Europe. The phytotoxin, viridiol, was previously isolated from culture extracts of H. pseudoalbidus and found to be toxic to leaves of F. excelsior. Thus, we were interested in learning to what extent viridiol is responsible for pathogenicity of H. pseudoalbidus and investigated this using twelve isolates of H. pseudoalbidus. We also included five isolates of the closely related avirulent species, Hymenoscyphus albidus, in our studies. Some, but not all, isolates of H. pseudoalbidus and H. albidus produced measurable quantities of viridiol in culture. Three tests were used to determine to what extent viridiol concentration correlates with virulence: culture extracts were tested for activity in leaf segment tests and for inhibition of germination of seedlings of Fraxinus excelsior; virulence of the isolates was tested following infection of axenically cultured ash seedlings. Activity of the culture extracts varied, as did virulence of the isolates following inoculation into seedlings. No correlations were found between viridiol concentration and activities of culture extracts in leaf segment tests or in the germination test, nor between viridiol concentration and disease symptoms when inoculated into seedlings. However, activities of culture extracts in leaf segment and in the germination test correlated, as did the results of each of these tests with virulence in the infection experiment. Apparently, as yet unidentified factors other than the concentration of viridiol play important roles in the virulence of H. pseudoalbidus.