Background: P. fluorescence is often recognized for their plant growth promoting activity as well as fungicidal activity against phytopathogens. In current study major aims was to observe the antifungal activity of P. fluorescence strains isolated from six farm soil samples of the Gazipur district area of Bangladesh and grown in different physicochemical parameters (pH, temperature, halotolerence, FeCl3 utilization, growth in different carbon and nitrogen sources) to observe the highest growth and formation of antifungal metabolites. Results: Products, grown in different physicochemical, were separated and purified to observe antimicrobial activity against phytopathogens. Six Pseudomonas fluorescence isolates were grown in progressive pH (5, 6, 7, 8 and 9), temperatures (4oC, 20oC, 37oC, 45oC and 55oC), salinity (1%, 3%, 5%, 10% and 15%) and FeCl3 concentrations (1%, 3%, and 5%) in King’s B broth medium and incubated at 37o C. Different substrate utilizing capabilities (C and N sources) and antimicrobial activities were also observed. Fraction chromatography was conducted to separate and purify the product. After 72 h of incubation, product yield was determined. All six isolates from different farm samples soils exhibited capability of utilizing different C and N sources. Isolates exhibited product yield from 2.1% up to 4.88%. Growth and product formation optimization exhibited different results with changing the parameters. The antimicrobial screening was conducted to detect antimicrobial activity of bacterial and fungal phytopathogens, isolated from different crops and fruits. P. fluorescence and its extracted material exhibited potential industrial applications as a fungicide. Conclusions: Optimizations of physical condition as well as capability of utilizing different N sources will have significant effect on the growth rate and production formation.