Aims: Present work describes the new enrichment method for the isolation of effective root colonizing and rhizospheric competent strains of genus fluorescent Pseudomonad and study of metabolic regulation of 2,4-DAPG biosynthesis in them under carbon sources and Pi levels. Methods and Results: Three rounds of plant assay was performed using root tip attached microorganism mixtures for the next round of root treatment followed by phonotypical separation of fluorescent colonies to isolate fluorescent pseudomonad strains from different crop and vegetables rhizospheres. Isolated strains were characterized for their Plant Growth Promoting Rhizobacteria (PGPR) traits viz phosphate solubilisation, production of siderophore, IAA, HCN, 1-aminocyclopropane-1-carboxylate /L-methionine utilization pathway and antifungal metabolites production. Isolated strains have shown high 2,4-diacetylphluoroglucinol production and strain G2 has shown 4.6 fold high production than Pf CHA0. Conclusions: Strain G1 and G8 supported 2,4-DAPG production under sucrose and found to be suitable biocontrol for sucrose rich rhizosphere. Strain G1 and G2 showed good 2,4-DAPG production at high Pi and will perform well in phosphate fertilizer supplemented soils. Significance of Study: Identification of factors favorable for bio-control will facilitate the targeted application of specific strains to plant rhizosphere/soil type/fertilizer supplemented suitable to their biocontrol activity i.e. “prescription” controls.