The rhizobacterial isolates from different agro ecological region of West Bengal, used in this study, were identified as fluorescent pseudomonads based on biochemical and molecular characterization. Six effective antagonist isolates were selected for further studies of secondary metabolite (siderophore) production with respect to bacterial growth at different time intervals (6 to 96 h), the nature of siderophore production (hydroxamate and/or catecholate type), effect of iron supplementation at its different concentration on siderophore production and antagonistic potentiality. Observations of higher level of growth and siderophore production, for most of the isolates, were recorded after 72 h of incubation. The maximum zone of inhibition and siderophore production was recorded at 0.5 µM and the least at 10 µM iron concentration. With the gradual increase in iron supplementation in king's B medium, the siderophore production as well as in vitro antagonistic potentiality decreased in terms of enhancement of diameter of fungal mycelium and reduction of percent inhibition of pathogen by fluorescent pseudomonads in dual culture assay method.