Recent studies have shown that dense yeast populations often occurring in floral nectar are numerically dominated by a few species from the flower-insect interface specialized genus Metschnikowia, while generalist yeast species commonly occurring on leaf surfaces, soil, freshwater, and air were rarely isolated from nectar samples. This study was designed to understand the main factors responsible for the assembly of nectar yeast communities, by combining field experiments with laboratory tests characterizing the physiological abilities of all yeast species forming the pool of potential colonizers for two Spanish flowering plants (Digitalis obscura and Atropa baetica). Yeast frequency and species richness were assessed in external sources (bee glossae, air, plant phylloplane) as well as in pollinator rewards (pollen, nectar). Yeasts were most frequent in external sources (air, flower-visiting insects), less so in the proximate floral environment (phylloplane), and least in pollen and nectar. Nectar communities appeared to be considerably impoverished versions of those in insect glossae and phylloplane. Nectar, pollen, and insect yeast assemblages differed in physiological characteristics from those in other substrates. Nectarivorous Metschnikowia were not more resistant than other yeast species to plant secondary compounds and high sugar concentrations typical of nectar, but their higher growth rates may be decisive for their dominance in ephemeral nectar communities.