Several hyphomycetes were recently isolated from asymptomatic or symptomatic vascular tissues of grapevines showing Petri disease symptoms in South Africa. In most cases, their status as pathogens was unknown and pathogenicity studies were, therefore, conducted to determine their potential as decline pathogens. The fungi included Acremonium cf. charticola, Acremonium cf. ochraceum, Cadophora luteo-olivacea, Phialemonium cf. curvatum, Pleurostomophora richardsiae, Phaeoacremonium (Pm.) krajdenii, Pm. parasiticum, Pm. subulatum, Pm. venezuelense and Pm. viticola. Of these, Pm. parasiticum and Pm. viticola have been associated with Petri disease symptoms, although their pathogenicity has not been tested. Phaeomoniella (Pa.) chlamydospora and Pm. aleophilum, known to be involved in Petri disease and esca, were included as positive controls. Pathogenicity studies were conducted in glasshouse experiments where grapevine rootstocks were artificially inoculated, as well as in the field. Data obtained after 3 months from a glasshouse trial were difficult to interpret, due to the small lesions and similarity in disease expression among different species. However, Pa. chlamydospora produced the largest lesions and was by far the most aggressive pathogen. To supplement the glasshouse trial, grapevine trunks and pruning wounds of Vitis vinifera cv. Periquita were artificially inoculated in the field. Field trials, rated after 14 months, confirmed Pa. chlamydospora to be the most aggressive pathogen, since it produced the largest trunk and pruning wound lesions. Furthermore, it was re-isolated more frequently than any of the other fungi, especially from the pruning wounds. All the fungi were able to infect, colonise and produce lesions statistically different to those caused by the water control and the non-pathogen in the field trial.