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Pseudomonas protegensPf-5 is an effective biocontrol agent that protects many crops against pathogens, including the fungal pathogenBotrytis cinereacausing gray mold disease inCannabis sativacrops. Previous studies have demonstrated the important role of antibiotics pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (DAPG) in Pf-5-mediated biocontrol. To assess the potential involvement of PLT and DAPG in the biocontrol exerted by Pf-5 againstB. cinereain the phyllosphere ofC. sativa, two knockout Pf-5 mutants were generated by in-frame deletion of genespltDorphlA, required for the synthesis of PLT or DAPG respectively, using a two-step allelic exchange method. Additionally, two complemented mutants were constructed by introducing a multicopy plasmid carrying the deleted gene into each deletion mutant.In vitroconfrontation assays revealed that deletion mutant ∆pltDinhibitedB. cinereagrowth significantly less than wild-type Pf-5, supporting antifungal activity of PLT. However, deletion mutant ∆phlAinhibited mycelial growth significantly more than the wild-type, hypothetically due to a co-regulation of PLT and DAPG biosynthesis pathways. Both complemented mutants recoveredin vitroinhibition levels similar to that of the wild-type. In subsequent growth chamber inoculation trials, characterization of gray mold disease symptoms on infected cannabis plants revealed that both ∆pltDand ∆phlAsignificantly lost a part of their biocontrol capabilities, achieving only 10 and 19% disease reduction respectively, compared to 40% achieved by inoculation with the wild-type. Finally, both complemented mutants recovered biocontrol capabilitiesin plantasimilar to that of the wild-type. These results indicate that intact biosynthesis pathways for production of PLT and DAPG are required for the optimal antagonistic activity ofP. protegensPf-5 againstB. cinereain the cannabis phyllosphere.