Two Pseudomonas aeruginosa isolates from natural biofilms (marine and freshwater environment) were investigated for exopolymer (EPS) production and chemical characterization. Both the isolates were categorized using molecular tools as P. aeruginosa species, The EPS production was distinct with various carbon and nitrogen sources, the average EPS yield by the two Pseudomonas strains was 55 µg ml-1. Modelling was done to establish the relation between observed and predicted EPS yields. The chemical composition, FTIR and Raman spectroscopy analysis of the two EPS showed that carbohydrate content was more in marine strain, while protein content was relatively high in the freshwater strain. Thermo-gravimetric analysis of the two EPS showed endothermic decomposition. Biochemical study by gel permeation chromatography showed that the marine strain EPS is a highly glycosylated biomolecule, while the freshwater EPS is a weakly glycosylated biomolecule with molecular weights 140,000 and 300,000 Daltons respectively. The EPS produced by the two Pseudomonas isolates has implication in process and chemical industries.