Organic fruit growing still depends heavily on the use of copper-based fungicides. One of the most prominent examples of destructive diseases is downy mildew of grapevine. To avoid the environmental impact of copper accumulation, sustainable alternatives for preventing downy mildew epidemics are needed, such as biological control agents (BCAs). Yeasts are fierce competitors in natural systems and represent promising candidates as BCAs. In this study, greenhouse trials were carried out to test the efficacy of three strains of yeast against P. viticola, namely, Pichia kluyveri A, P. kluyveri B, and Saccharomyces cerevisiae S101. A novel leaf-fertiliser Aqua-Hort® was also tested. This contains a copper microdose that could reduce pollution with copper. All yeasts and the copper treatment reduced disease severity of downy mildew significantly when applied once before inoculation with P. viticola sporangia. The highest efficacy was obtained with copper. Moreover, application of P. kluyveri B reduced the number of indigenous fungi in the phyllosphere of grapevines significantly, compared to S. cerevisiae. The results indicate fungal interactions and a putative killer activity by P. kluyveri. Conclusively, indigenous grapevine yeast showed potential as a BCA against P. viticola.