Cotton seedling disease caused by Pythium ultimum Trow and Rhizoctonia solani Kühn occurs worldwide in soils ranging from pH 4.5 to 8.5. Studies with cotton have not yet established the relative importance of two classes of secondary metabolites synthesized by soilborne pseudomonads, siderophores (sid) (low molecular weight Fe⁺³ chelators) and antibiotics (ant), in the suppression of these pathogens. Greenhouse bioassays to screen for rhizosphere competent strains identified a single strain of Pseudomonas chlororaphis (L-850), that produced siderophores and multiple antifungal antibiotics, including one or more phenazines. A Tiff Image Analyzer (TIA) software program was developed that allowed assessment of wild-type (wt) L-850, and (ant) and (sid) mutant populations as a function of cotton root surface area (cm²) in the absence of soil irrigation. Bacterial density and distribution patterns on roots evaluated 22, 36, and 50 DAP, in two pathogen-free soils (pH 5.7, high Fe⁺³, high phosphorus (P); pH 8.0, low Fe⁺³, low P) indicated that populations of both wt and mutants persisted after day 22 at levels between log 4.6 (lower laterals) to log 6 cfu/cm² (upper tap) even as total root area increased 122% from day 22 to 50. Population densities of all strains were consistently 1/8 to 3/4 log unit lower in the pH 5.7 soil on the lower tap and upper lateral roots, respectively. The loss of siderophore production appeared to enhance the rhizosphere competence of strain L-850. For greenhouse trials with three pathogen inoculum densities (low, intermediate, high) protection against preand postemergence damping-off (phase 1) and hypocotyl/root rot of young plants (phase 2) by the (sid) and wt strains was similar (P = 0.05) whereas, protection by seed treatment with the (ant) mutant was reduced. The level of suppression provided by L-850 was equivalent (P = 0.05) to the standard fungicide at low and intermediate pathogen pressure. These studies demonstrated a minimum contribution of siderophores in the biological control of cotton seedling disease and established a significant role for antibiotic biosynthesis over a range of soil physical and chemical characteristics.