Antibiotics are generally considered as low molecular weight organic compounds produced by microbes and play an active role in the bio-control of plant diseases. Fluorescent pseudomonad bacterial strains are known to suppress fungal growth under in vitro as well in vivo by the production of one or more antifungal antibiotics. Production of antibiotic compounds by different age old liquid formulations of P. fluorescens (Pf1) was assayed by Thin Layer Chromatography (TLC). The presence of the antimicrobial compounds viz., Phenazine 1-carboxylic acid (PCA), Pyoluteorin (PLT), Oxychlororaphine, Pyoluteorin (PLT), Pyocyanine were detected with different Rf values. 180 days old Pf1 liquid formulation was verified for the production of diacetylphloroglucinol (DAPG) by using Analytical High Performance Liquid chromatography (HPLC). The result of HPLC analysis revealed that DAPG from Pf1 was recorded the retention time of 4.1 min at 270 nm. Similarly same result was obtained from the DAPG standard with the retention time of 4.5 min. Hence the present study, indicate that different days old formulation of Pf1 up to180 days showed positive result for the production of all these antibiotics. It provides the evidence to Pf1 liquid formulation might act against the plant disease through the mechanism of antibiosis when applied to cropping system. Therefore, antibiotics improve the ecological fitness of these bacteria in the liquid culture which can further influence long-term biocontrol efficacy and supports the use liquid based biocontrol agents for sustainable production of agricultural crops.