Pseudozyma flocculosa is an epiphytic yeast with powerful antagonistic activity against powdery mildews. This activity has been associated with the production of a rare antifungal glycolipid, flocculosin. In spite of the discovery of a specific gene cluster for flocculosin synthesis, attempts to ascribe a functional role to the molecule have been hampered by the inability to efficiently transform P. flocculosa. In this study, two different approaches, target gene replacement by homologous recombination (HR) and CRISPR-Cas9 based genome-editing, were utilized to decipher the role of flocculosin in the biocontrol activity of P.flocculosa. It was possible to alter the production of flocculosin through edition of fat1 by HR, but such mutants displayed abnormal phenotypes and the inability to produce sporidia. Sequencing analyses revealed that transformation by HR led to multiple insertions in the genome explaining the pleiotrophic effects of the approach. On the other hand, CRISPR-Cas9 transformation yielded one mutant that was altered specifically in the proper synthesis of flocculosin. Notwithstanding the loss of flocculosin production, such mutant was phenotypically similar to the wild-type, and when tested for its biocontrol activity against powdery mildew, displayed the same efficacy. These results offer strong evidence that flocculosin-mediated antibiosis is not responsible for the mode of action of P. flocculosa and highlight the potential of CRISPR-Cas9 for functional studies of otherwise difficult-to-transform fungi such as P. flocculosa.