Fourier Transform Infrared (FTIR) technique is a valuable tool for investigation of biochemical composition of bacterial biofilms.Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy was used to analyze and compare Streptococcus agalactiae bio film exopolysaccharide (EPS) matrix and free cell components. As per the standardization of growth kinetics and biofilm formation ofS.agalactiaein our earlier studies, biofilm was grown in 0.625 per cent Luria-Bertani (LB) broth with one per cent glucose on 0.3 per cent bentonite clay as inert surface for 3 days and 2.5 per cent LB glucose on 0.5 mm glass beads as inert surface for 48 hrs. Free cells were grown in 2.5 per cent LB glucose for 24 hrs. Biofilm EPS and free cell componenets were subjected for ATR-FTIR spectroscopy. The results revealed marked differences in the chemical composition of the biofilm EPS and free cell components. Differences were observed particularly in the carbohydrate region between 1200‐900 cm‐1 wavenumber and the protein region. Interestingly, the S. agalactiae biofilms grown on bentonite clay and glass beads showed increasing IR signalling intensities at the polysaccharide spectral regions, instead, the free cells showed decreasing IR signalling intensities at this region, whereas free cells revealed prominent peaks of proteins. This finding clearly indicated that the polysaccharide concentrations were more in biofilms compared to free cell components. These temporal differences reflect the presence of excess amount of polysaccharides in biofilms and may be related to exopolysacharide production during biofilm development.This is the first report of S. agalactiae biofilm analysis using FTIR spectroscopy.