Pseudomonas fluorescens is a diverse bacterial species known for its ubiquity in natural habitats and the production of structurally diverse, bioactive secondary metabolites. The high degree of ecological and metabolic diversity represented in P. fluorescens is reflected in the genomic diversity displayed among strains. Certain strains, such as the biological control bacterium P. fluorescens Pf-5, live in association with plants, protecting them from infection by plant pathogens. Strain Pf-5 produces an array of structurally-characterized secondary metabolites that are toxic to plant pathogenic bacteria, fungi and Oomycetes. Biosynthetic gene clusters for many of these metabolites are located in lineage-specific regions absent from the genomes of other strains of P. fluorescens. Orphan gene clusters, which encode for the biosynthesis of unknown natural products, have also been identified in lineage-specific regions of the Pf-5 genome and the following products identified through combined bioinformatic and chemical analyses. The novel cyclic lipopeptide orfamide A lyses zoospores produced by phytopathogenic Phytophthora spp. The FitD insect toxin contributes to the newly-discovered insecticidal activity of Pf-5, and several analogs of rhizoxin, a macrocyclic lactone, exhibit antifungal activity. Recently, orphan gene clusters expressed under the control of global regulators such as GacA have been identified via transcriptome analysis of Pf-5, demonstrating the value of global-regulator-based genome mining as an approach to decipher the secondary metabolome of Pseudomonas spp. We are also employing microarrays to gain a holistic view of genome expression profiles of Pf-5 living on seed surfaces, the environment where the bacterium interacts with seed-infecting fungi and Oomycetes to affect biocontrol. A series of Pf-5 mutants having deletions in one or many (up to seven) known or orphan gene clusters have been derived and are being tested in a series of bioassays. These approaches are providing new insights into the metabolic capacity of this bacterium, its activity on plant surfaces, and its interactions with plants, insects and other microorganisms.