Antagonistic rhizobacteria can act on plant pathogenic fungi through antibiosis, fungistasis, or induction of a plant defense response. To explore different biocontrol mechanisms, an in vitro confrontation assay was conducted with the rhizobacterium Pseudomonas fluorescens Pf29Arp as a biocontrol agent of the fungus Gaeumannomyces graminis var. tritici (Ggt) on wheat roots. During the 10-day infection process, the disease extension, the bacterial and fungal root colonization rates, and the transcript levels of candidate fungal pathogenicity genes were monitored. The development of the Ggt-induced disease (attack frequency and necrosis length) was reduced in Pf29Arp-inoculated wheat roots. Throughout the interactions, the growth rates of Pf29Arp increased and the bacterial antagonism and colonization had no significant effect on root colonization by Ggt. The expression of fungal genes was quantified in planta by quantitative reverse transcription-polymerase chain reaction and an innovative universal reference system. During the early stages of the tripartite interaction, several of the fungal genes assayed were down-regulated by Pf29Arp, including two laccases, a β-1, 3-exoglucanase, and a mitogen-activated protein kinase. We conclude that Pf29Arp antagonism acts through the alteration of fungal pathogenesis.