The recently characterized strain Pseudomonas orientalis F9, an isolate from apple flowers in a Swiss orchard, exhibits antagonistic traits against phytopathogens. At high colonization densities, it exhibits phytotoxicity against apple flowers. P. orientalis F9 harbors biosynthesis genes for the siderophore pyoverdine as well as for the antibiotics safracin and phenazine. To elucidate the role of the three compounds in biocontrol, we screened a large random knockout library of P. orientalis F9 strains for lack of pyoverdine production or in vitro antagonism. Transposon mutants that lacked the ability for fluorescence carried transposons in pyoverdine production genes. Mutants unable to antagonize Erwinia amylovora in an in vitro double-layer assay carried transposon insertions in the safracin gene cluster. As no phenazine transposon mutant could be identified using the chosen selection criteria, we constructed a site-directed deletion mutant. Pyoverdine-, safracin-, and phenazine mutants were tested for their abilities to counteract the fire blight pathogen Erwinia amylovora ex vivo on apple flowers or the soilborne pathogen Pythium ultimum in vivo in a soil microcosm. In contrast to some in vitro assays, ex vivo and in vivo assays did not reveal significant differences between parental and mutant strains in their antagonistic activities. This suggests that, ex vivo and in vivo, other factors, such as competition for resources or space, are more important than the tested antibiotics or pyoverdine for successful antagonism of P. orientalis F9 against phytopathogens in the performed assays.