The Pseudomonas fluorescens isolate Pfl was found to inhibit the growth of pathogen Alternaria palandui, in vitro. In the present study, foliar application of a talc-based formulation of Pfl significantly reduced the incidence of leaf blight of onion, caused by A. palandui. Induction of defense-related proteins viz., chitinase, beta-1,3 glucanase, peroxidase (PO) and polyphenol oxidase (PPO) by application of Pfl, was studied against A. palandui infection in resistant (IHR 56) and susceptible (MDUI) onion cultivars. Chitinase in both cultivars, with or without challenge-inoculation of A. palandui revealed changes in the isoform pattern. The Native-PAGE of PO showed induction of PO2 isoform in both the cultivars, in response to inoculation of pathogen. Isoform analysis of PPO also exhibited induction in the Pfl-treated plants challenged with pathogen. Similarly, the activity of beta-1,3-glucanase was greatly induced in Pfl-treated plants, challenged with pathogen as compared to controls. Thus, the P. fluorescens-treated plants showed significant increase in the levels of the defense enzymes, in comparison to the plants challenged with the pathogen.