The O-antigen determines the specificity of bacterial serotype, a sort of bacterial fingerprinting. In this work we report the extraction, purification and characterization of the O-antigen of two pathogenic bacteria, Escherichia coli O16 and Salmonella typhimurium. Molecular fingerprints found in the vibrational spectra represent a powerful analytical technique for identification (or differentiation) of molecular moieties in complex systems such as pathogens. In addition, advantages of vibrational Raman scattering are unique thanks to the high sensitivity and specificity achieved via surface-enhanced Raman scattering (SERS). SERS is used here to take advantage of characteristic vibrational frequency differences of O-antigens, thus allowing bacterial differentiation. Characteristic fundamental vibrational modes associated with the monosaccharide N-acetylglucosamine and deformations of the O-antigen chains provide the main spectroscopic differences between the O-antigens of E. coli O16 and S.typhimurium.