A collection of yeasts isolated from nectar of flowers of Protea caffra (Proteaceae) and associated scarab beetles (Atrichelaphinis tigrina, Cyrtothyrea marginalis, Trichostetha fascicularis and Heterochelus sp.) and drosophilid flies in South Africa, contained 28 isolates that could not be assigned to known species. Comparisons of the D1/D2 domains of the large subunit rRNA gene demonstrated the existence of three separate phylotypes with an affinity to the genus Metschnikowia and more specifically to the beetle-associated large-spored Metschnikowia clade. Twenty-six strains that had similar D1/D2 sequences were mixed in all pairwise combinations. They were found to mate and give rise to large asci typical of those in the clade. The name Metschnikowia proteae sp. nov. (type strain EBDT1Y1T = CBS 12522T = NRRL Y-48784T; allotype strain EBDC2Y2 = CBS 12521 = NRRL Y-48785) is proposed to accommodate this novel species. The ecology of this novel yeast species is discussed in relation to its potential plant and insect host species. The additional two single strains isolated from Heterochelus sp. represent two novel undescribed species (Candida sp. 1 EBDM2Y3 and Candida sp. 2 EBDM8Y1). As these single strains are probably haploid mating types of Metschnikowia species, their description is deferred until the species are sufficiently well sampled to permit meaningful descriptions.