Species of Ophiostoma include some of the world's best known fungal pathogens of trees. Most members are vectored by arthropods. One of the most unusual niches in which Ophiostoma species have been found is within the infructescences of Protea species in South Africa. Recent molecular phylogenetic reconstructions on Ophiostoma s.l. suggested that the three Ophiostoma spp. specifically found in Protea infructescences, form a strongly supported monophyletic lineage within Ophiostoma s.s. In this study, new collections of Ophiostoma from Protea infructescences were subjected to molecular phylogenetic reconstructions based on large subunit, ITS and beta-tubulin sequence data. Using these techniques, at least five undescribed species of Ophiostoma have been identified from these plants. Intriguingly, our results also suggest a polyphyletic origin for the Protea-associated Ophiostoma spp. This indicates multiple invasions of this unusual niche, by these fungi. Our results also revealed the first case of an Ophiostoma sp. jumping hosts between a native Protea sp. and the non-native tree genus Eucalyptus. The second aim of this study was to identify putative vectors of the Ophiostoma spp. inhabiting Protea infructescences using both molecular and direct isolation methods. The presence of reproductive propagules of Ophiostoma spp. was confirmed on four Protea-associated mite species (Oodinychus sp., two Tarsonemus spp. and Proctolaelaps vandenbergi) at high frequencies. The Oodinychus sp. mite showed significantly higher reproductive rates when fed exclusively on Ophiostoma splendens than when it was fed on various other fungi. This suggests a mutualistic association between the Oodinychus sp. and O. splendens. Long distance dispersal of these mites was restricted to vectored dispersal via Protea-infructescence inhabiting beetles (e.g. Genuchus hottentottus). Mites collected from these beetles were found to vector spores of various Ophiostoma spp. Based on these results, our view is that these mites act as primary vectors of the Ophiostoma spp. in Protea infructescences.