Pseudomonas fluorescens is one of plant Growth Promoting Rhizobacteria (PGPR) members which has a major role in the biological control of bacterial and fungal pathogens. A research was conducted at the Laboratory of Microbiology, Faculty of Agriculture, Ain Shams University, Qalubyia governorate intended for isolating Pseudomonas fluorescens isolate, efficient in antibiotic production. For isolation, soil samples, collected from Faculty of agriculture farm soil at depth of 10 cm, were screened for Ps. fluorescens isolates by cultivating the samples on King’s medium. Out of 30 isolates obtained, one was selected based on its high lipid content, because of the lipid’s correlation to antibiotic production and inhibitory activity. The selected isolate was characterized by morphological, physiological, biochemical tests then confirmed its identity by 16S rRNA gene sequence analysis and named Pseudomonas fluorescens NBRC-14160 16srRNA. Morphological features showed that Ps. fluorescens NBRC-14160 has large colonies, with irregular surface, opaque, producing green fluorescent pigments. Cells are short rods, Gram negative. Physiological features indicated that Ps. fluorescens NBRC-14160 is capable of producing several exoenzymes including lipase, phospholipase, protease and chitinase and incapable of producing amylase and cellulase. Carbohydrate fermentation tests were positive for fructose, glucose, D-glucose, and galactose, forming acids after 24 hrs. of incubation at 30˚C. However, it loses the ability to ferment inositol, mannose, xylose, mannitol, raffinose and rhamnose sugars. It’s lipid content was 607 mg/g. Inhibitory activity was studied by the method of disc diffusion test against nine pathogenic bacterial and fungal strains. Staphylococcus aureus was the most sensitive bacterial pathogen towards P. fluorescens NBRC-14160 with a 4.5 cm zone of inhibition, while Serratia marscens had 0.95 cm zone. Aspergillus niger and Alternaria solani were the most sensitive fungal pathogens towards P. fluorescens NBRC-14160 with 2.55 and 2.5 cm of inhibition zone, respectively. However, the most resistant fungus was Fusarium oxysporum with 1.3 cm inhibition zone.