BackgroundPseudozyma flocculosais a highly efficient biocontrol agent (BCA) of powdery mildews whose mode of action remains elusive. It is known to secrete unique effectors during its interaction with powdery mildews but effectors have never been shown to be part of the arsenal of a BCA. Here, we characterize the role of the effector Pf2826 released byPseudozyma flocculosaduring its tripartite interaction with barley and the pathogen fungusBlumeria graminisf. sp.hordei.ResultsWe utilized CRISPR-Cas9-based genome editing and confirmed that secretedP. flocculosaeffector Pf2826 is required for full biocontrol activity. We monitored the localization of the effector Pf2826 with C-terminal mCherry tag and found it localized around the haustoria and on powdery mildew spores. His-tagged Pf2826 recombinant protein was expressed, purified, and used as bait in a pull-down assay from total proteins extracted during the tripartite interaction. Potential interactors were identified by LC–MS/MS analysis after removing unspecific interactions found in the negative controls. A two-way yeast two-hybrid assay validated that Pf2826 interacted with barley pathogenesis-related (PR) proteins HvPR1a and chitinase and with an effector protein from powdery mildew.ConclusionsIn contrast to the usual modes of action of competition, parasitism, and antibiosis ascribed to BCAs, this study shows that effector pf2826 plays a vital role in the biocontrol activity ofP. flocculosaby interacting with plant PR proteins and a powdery mildew effector, altering the host–pathogen interaction.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12915-023-01624-z.