Pseudomonas fluorescens F113 is an effective biocontrol agent against Pythium ultimum, the causative agent of damping-off of sugarbeet seedlings. Biocontrol is mediated via the production of the anti-fungal metabolite 2,4-diacetylphloroglucinol (Phl). A genetic approach was used to further enhance the biocontrol ability of F113. Two genetically modified (GM) strains, P. fluorescens F113Rif (pCU8.3) and P. fluorescens F113Rif (pCUP9), were developed for enhanced Phl production and assessed for biocontrol efficacy and impact on sugarbeet in microcosm experiments. The multicopy plasmid pCU8.3 contains the biosynthetic genes (phlA, C, B and D) and the putative permease gene (phlE) of F113 cloned into the rhizosphere stable plasmid pME6010, independent of external promoters. The plasmid pCUP9 consists of the Phl biosynthetic genes cloned downstream of the constitutive Plac promoter in pBBR1MCS. Introduction of pCU8.3 and pCUP9 into P. fluorescens F113 significantly altered the kinetics of Phl biosynthesis when grown in SA medium. A significant and substantial increase in Phl production by the GM strains was observed in the early logarithmic phase and stationary phase of growth compared with the wild-type strain. In microcosm, the two Phl overproducing strains proved to be as effective at controlling damping-off disease as the proprietary fungicide treatment, indicating the potential of genetic modification for plant disease control.